Anti-cocaine compositions and treatment

ABSTRACT

Embodiments of the invention disclosed herein generally relate to anti-cocaine therapeutics. Specifically, some embodiments of the invention relate to highly efficient, thermostable, and long-lasting cocaine esterase (CocE) mutants that can protect against the toxic and reinforcing effects of cocaine in subjects. Provided herein are mutant CocE polypeptides displaying thermostable esterase activity. Also provided are methods of treating cocaine-induced conditions in a subject in need via administration of mutant CocE as well as methods for high-throughput screening of candidate esterase polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. NonprovisionalApplication Ser. No. 12/373,510, filed 10 Jul. 2007 issued as U.S. Pat.No. 8,318,158 on 27 Nov. 2012; which claims the benefit of priority toInternational Patent Application No. PCT/US07/15762, filed 10 Jul. 2007,and U.S. Provisional Application No. 60/819,569, filed 10 Jul. 2006;each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant DA021416 andGrant DA013683 awarded by National Institute of Health. The governmenthas certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

The Sequence Listing, which is a part of the present disclosure,includes a computer readable form and a written sequence listingcomprising nucleotide and/or amino acid sequences of the presentinvention. The sequence listing information recorded in computerreadable form is identical to the written sequence listing. The subjectmatter of the Sequence Listing is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The invention disclosed herein generally relates to anti-cocainetherapeutics.

BACKGROUND

Abuse of cocaine is an intractable social and medical problem that isresistant to remediation through pharmacotherapy. Cocaine acts to blockthe reuptake of monoamines, dopamine, norepinephrine, and serotin thusprolonging and magnifying the effects of these neurotransmitters in thecentral nervous system (Benowitz N L (1993) Pharmacol Toxicol 72, 3-12).Cocaine toxicity is marked by both convulsions and cardiac dysfunction(e.g., myocardial infarction, cardiac arrhythmias, increased bloodpressure, stroke, or dissecting aneurysm, and increased myocardialoxygen demand), due to effects on neurotransmitter systems andmyocardial sodium channel blockade (Bauman J L and DiDomenico R J (2002)J Cardiovasc Pharmacol Ther 7, 195-202; Wilson L D and Shelat C (2003) JToxicol Clin Toxicol 41, 777-788; Knuepfer M M (2003) Pharmacol Ther 97,181-222). Because of cocaine's ability to readily cross the blood brainbarrier and its widespread effects on the central and peripheral nervoussystems, overdose can result in sudden death (see Bauman J L andDiDomenico R J (2002) J Cardiovasc Pharmacol Ther 7, 195-202, forreview).

Although the mechanism of cocaine's action is well understood, thisinformation has not yet resulted in the development of an effectiveantagonist of cocaine that could be used in abuse and overdosesituations. The rapid and pleiotropic effects of cocaine present acomplex problem for the treatment of acute cocaine toxicity (Carroll FI, Howell L L and Kuhar M. J (1999) J Med Chem 42, 2721-2736). The twotypes of therapies that are available for the treatment of opioid abuse,antagonism (e.g., naltrexone) and replacement (e.g., methadone), do nothave parallels in the case of cocaine, although attempts at the latterare being considered (e.g., J. Grabowski et al. (2004) AddictiveBehaviors 29, 1439-1464). One approach is to prevent or reduce thecocaine from reaching sites of action by administering either endogenousesterases, cocaine specific antibodies, or a catalytic antibody.

Naturally occurring cocaine is hydrolyzed at the benzoyl ester by serumbutyrylcholinesterase (BChE) to nontoxic ecgonine methyl ester andbenzoic acid. In the liver, carboxylesterase hCE-2 hydrolyzes the methylester to yield benzoylecgonine and methanol (see e.g., FIG. 1). Theelimination half-life of cocaine in the blood ranges from 0.5 to 1.5 hr(T. Inaba (1989) Canadian Journal of Physiology & Pharmacology 67,1154-1157). There have been a few attempts to use naturally occurringBChE or genetically engineered BChE to increase cocaine breakdown (seee.g., Carmona et al. (2000) Drug Metabolism & Disposition 28, 367-371;Xie et al. (1999) Molecular Pharmacology 55, 83-91; Sun et al. (2002a)Molecular Pharmacology; Sun et al. (2002b) Pharmacology & ExperimentalTherapeutics 302, 710-716; Duysen et al. (2002) Journal of Pharmacology& Experimental Therapeutics 302, 751-758; Gao Y and Brimijoin S (2004)Journal of Pharmacology & Experimental Therapeutics 310, 1046-1052; Gaoet al. (2005) Molecular Pharmacology 67, 204-211). Other researchershave utilized a monoclonal antibody, Mab 15A10, as a catalytic antibodyto cocaine (see e.g., Landry et al, 1993; Mets et al., 1998; Baird etal., 2000; Larsen et al., 2004), while others are exploring the use ofcocaine vaccines (see e.g., Kosten et al. (2002) Vaccine 20, 1196-1204).

TABLE 1 Kinetics of several cocaine hydrolyzing enzymes against (-)cocaine. Kcat Km Efficiency Enzyme (min−1) (μM) (kcat/Km) Reference BChE4.1 4.5 9.1 × 10⁶ Sun et al., 2002a Ala328W/Y332A 154 18 8.5 × 10⁶ Sunet al., 2002a Mab15A10 2.2 220  1 × 10⁴ Larsen et al., 2004 AME 359 62020 3.1 × 10⁷ Gao et al., 2005 CocE 468 0.64 7.2 × 10⁸ Turner et al.,2002

A bacterium, Rhodococcus sp. MB 1, indigenous to the soil surroundingthe coca plant, has evolved the capacity to utilize cocaine as its solecarbon and nitrogen source. The bacterium expresses a cocaine esterase(CocE) that acts similarly to BChE to hydrolyze the benzoyl ester ofcocaine, yielding ecgonine methyl ester and benzoic acid (see e.g.,FIG. 1) (Bresler et al. (2000) Appl Environ Microbiol 66, 904-908;Turner et at. (2002) Biochemistry 41, 12297-12307; Larsen et al. (2002)Nature Struct Biol 9, 17-21). The gene for CocE has been isolated andcloned (Bresler et al. (2000) Appl Environ Microbiol 66, 904-908), andthe crystal structure of CocE has been determined (Turner et at. (2002)Biochemistry 41, 12297-12307; Larsen et al. (2002) Nature Struct Biol 9,17-21). The structure of CocE (see e.g., FIG. 2) reveals a classicserine esterase fold in addition to two other domains that combine toform a cocaine binding pocket. Altering any of three amino acids (Asp,His, or Ser) within the catalytic triad in the active site (for review,see Dodson G and Wlodawer A (1998) Trends Biochem Sci 23, 347-352)inactivates the esterase activity against cocaine. Furthermore, mutationof residues that make contact with the benzoate moiety of cocaine (e.g.,Tyr44) also disrupts cocaine hydrolysis, presumably through impairingoxyanion stabilization in the transition state (Turner et al. (2002)Biochemistry 41, 12297-12307; Larsen et al. (2002) Nature StructuralBiology 9, 17-21). The purified enzyme (MW ˜65 kDa) catalyzes cocainevery efficiently with Michaelis-Menten kinetics k_(cal)=7.2 s⁻¹ andKm=640 nM (Turner et al. (2002) Biochemistry 41, 12297-12307; Larsen etal. (2002) Nature Structural Biology 9, 17-21), nearly three orders ofmagnitude greater than endogenous esterases and, most likely, would actquickly enough to detoxify humans who have overdosed on cocaine (Landryet al. (1993) Science 259, 1899-1901; Mets et al. (1998) NationalAcademy of Sciences of the United States of America 95, 10176-10181).Additionally, the esterase also metabolizes cocaethylene, a potentmetabolite of cocaine and alcohol, almost as efficiently as itmetabolizes cocaine (k_(cat)=9.4 s⁻¹ and Km=1600 nM) (Turner et al.(2002) Biochemistry 41, 12297-12307; Larsen et al. (2002) NatureStructural Biology 9, 17-21).

Thus, it would be desirable to provide a stable CocE for anti-cocainetherapeutics.

SUMMARY OF THE INVENTION

Accordingly, the present inventors have succeeded in discovering highlyefficient, thermostable, and long-lasting cocaine esterase mutants thatcan protect against the toxic and reinforcing effects of cocaine insubjects.

One aspect of the invention provides isolated mutant cocaine esterase(CocE) polypeptides of the wild-type CocE amino acid sequence (e.g., SEQID NO: 1) but with at least one amino acid residue substituted. Themutant CocE polypeptides have esterase activity with increasedthermostability at 37° C. as compared to wild-type CocE.

Various embodiments include mutant CocE polypeptides with at least two,three, four, five, or more substitutions of the wild-type CocE aminoacid sequence. Examples of mutant CocE polypeptides within the scope ofthe invention include those with an amino acid sequence of SEQ ID NO: 3(L163V); SEQ ID NO: 7 (V225I); SEQ ID NO: 8 (I218L); SEQ ID NO: 9(A310D); SEQ ID NO: 10 (A149S); SEQ ID NO: 11 (S159A); SEQ ID NO: 12(S265A); SEQ ID NO: 13 (S56G); SEQ ID NO: 14 (W220A); SEQ ID NO: 16(S140A); SEQ ID NO: 17 (F189L); SEQ ID NO: 18 (A193D); SEQ ID NO: 19(T254R); SEQ ID NO: 20 (N42V); SEQ ID NO: 21 (V262L); SEQ ID NO: 22(L508G); SEQ ID NO: 23 (Y152H); SEQ ID NO: 24 (V160A); SEQ ID NO: 25(T172R); SEQ ID NO: 26 (Y532F); SEQ ID NO: 27 (T74S); SEQ ID NO: 28(W285T); SEQ ID NO: 29 (L146P); SEQ ID NO: 30 (D533S); SEQ ID NO: 31(A194R); SEQ ID NO: 32 (G173Q); SEQ ID NO: 33 (C477T); SEQ ID NO: 34(K531A); SEQ ID NO: 35 (R41I); SEQ ID NO: 36 (L119A); SEQ ID NO: 37(K46A); SEQ ID NO: 38 (F84Y), T172R-G173Q (SEQ ID NO: 39); L169K (SEQ IDNO: 40); F189A (SEQ ID NO: 41), N197K (SEQ ID NO: 42), R182K (SEQ ID NO:43), F189K (SEQ ID NO: 44), V190K (SEQ ID NO: 45), Q191K (SEQ ID NO:46), and A194K (SEQ ID NO: 47), or a functional fragment(s) thereof.Additional exemplary mutant CocE polypeptides include, F189A/T172R,T172R/A193D, T172R/G173Q-1175-G-G-A186, T172R/G173Q-T176-G-G-D185, andthe like. Given the naming conventions and polypeptide sequencesdisclosed herein, one skilled in the art could determine the polypeptidesequences for the above-named mutant CocE polypeptides.

Another aspect of the invention provides pharmaceutical compositionsthat include among their components a mutant CocE polypeptide within thescope of the invention and a pharmaceutically acceptable carrier orexcipient.

Another aspect of the invention provides isolated nucleic acids encodingthe mutant CocE polypeptide described herein. In various embodiments,the nucleic acids include those with sequences that hybridize to thenucleic acid encoding wild-type CocE (e.g., SEQ ID NO: 2), or thecomplement thereto, under high stringency conditions. Such isolatednucleic acid encodes a mutant CocE polypeptide having esterase activitywith increased thermostability at 37° C. as compared to wild-type CocE.Various embodiments of the isolated nucleic acid sequence have at leastabout 85% sequence identity with the nucleic acid sequence of wild-typeCocE (e.g., SEQ ID NO: 2). For example, the isolated nucleic acidsequence has at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, and 99% sequence identity of wild-type CocE (e.g.,SEQ ID NO: 2).

In various embodiments, mutant CocE polypeptides (or polypeptidesencoded by nucleic acids within the scope of the invention) haveincreased melting temperatures as compared to wild-type CocE. In variousembodiments, mutant CocE polypeptides (or polypeptides encoded bynucleic acids within the scope of the invention) increase mutant CocEpolypeptide thermostability over wild-type CocE by at least about 2.0kcal/mol. For example, the increased thermostability can be at leastabout 2.1 kcal/mol, at least about 2.2 kcal/mol, at least about 2.3kcal/mol, at least about 2.4 kcal/mol, at least about 2.5 kcal/mol, atleast about 2.6 kcal/mol, at least about 2.7 kcal/mol, at least about2.8 kcal/mol, at least about 2.9 kcal/mol, at least about 3.0 kcal/mol,at least about 3.1 kcal/mol, at least about 3.2 kcal/mol, at least about3.3 kcal/mol, at least about 3.4 kcal/mol, at least about 3.5 kcal/mol,at least about 3.6 kcal/mol, at least about 3.7 kcal/mol, at least about3.8 kcal/mol, at least about 3.9 kcal/mol, at least about 4.0 kcal/mol,at least about 4.1 kcal/mol, at least about 4.2 kcal/mol, at least about4.3 kcal/mol, at least about 4.4 kcal/mol, or at least about 4.5kcal/mol.

In various embodiments, mutant CocE polypeptides (or polypeptidesencoded by nucleic acids within the scope of the invention) have reducedimmunogenicity as compared to wild-type CocE.

In some embodiments, the thermostable mutant CocE polypeptides have lessesterase activity than wild-type CocE. For example, thermostable CocEmutants can have about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 99% of the esterase activity of wild-type CocE.In other embodiments, the mutant CocE polypeptides have approximatelythe same, or greater, catalytic efficiency of wild-type CocEpolypeptides. For example, thermostable CocE mutants can have about100%, about 110%, about 120%, about 130%, about 140%, about 150%, ormore of the esterase activity of wild-type CocE.

In various embodiments, the mutant CocE polypeptides are pegylated. Invarious embodiments, the mutant CocE polypeptide is encapsulated in ared blood cell. For example, a pegylated mutant CocE polypeptide(s) (ora pharmaceutical composition including a pegylated mutant CocEpolypeptide(s)) can be encapsulated in a RBC.

In some embodiments, the mutant CocE polypeptides are stabilized by asubstrate, product, and/or inhibitor.

Another aspect of the invention provides for methods of treating acocaine-induced condition. In such methods, a therapeutically effectiveamount of a mutant CocE polypeptide(s) (or a pharmaceutical compositionincluding a mutant CocE polypeptide(s)) within the scope of theinvention is administered to a subject in need thereof. In variousembodiments, the cocaine-induced condition includes cocaine overdose,cocaine toxicity, cocaine addiction, cocaine dependence, and/or somecombination thereof.

Yet another aspect of the invention provides for a high throughputscreening method for identification of thermostable mutant CocEpolypeptides. In such screening method, a cell is stably transformedwith a nucleic acid encoding a mutant CocE polypeptide candidate. Themutant CocE polypeptide is expressed in the cell. The expressed mutantCocE polypeptide is isolated or displayed. The esterase activity of theisolated mutant CocE polypeptide is measured at one or more temperaturesto determine thermostability of the isolated mutant CocE polypeptides.Such temperature(s) can be from about 30° C. to about 50° C. Mutant CocEpolypeptide with esterase activity at the predetermined temperature(s)are selected.

In some embodiments of the screening method, measuring esterase activityof the isolated mutant polypeptides can be accomplished by contactingthe isolated mutant CocE polypeptide with (i) cocaine and a pH indicatoror (ii) a thio-derivative of cocaine and a thiol indicator. Any changein the pH indicator or the thiol indicator is then detected. Such achange is correlated with the formation of benzoic acid from thehydrolysis of cocaine or cocaine derivative by the mutant CocEpolypeptide.

Some embodiments of the screening method further include conductingseveral cycles of the screening procedure at increasing temperatures formeasuring esterase activity. For example, the first cycle can employ atemperature for measuring esterase activity of about 30° C. while asubsequent cycle can employ a temperature for measuring esteraseactivity of about 45° C.

In some embodiments of the screening method, expression of mutant CocEpolypeptide occurs at a temperature at which wild type CocEsubstantially retains catalytic activity. In other embodiments of thescreening method, expression of mutant CocE polypeptide occurs at atemperature at which wild-type CocE polypeptide substantially partitionsinto inclusion bodies. For example, the expression temperature can be atleast about 35° C., at least about 36° C., at least about 37° C., atleast about 38° C., at least about 39° C., or at least about 40° C.

Other objects and features will be in part apparent and in part pointedout hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 is a diagram illustrating the metabolism of cocaine by variousenzymatic reactions.

FIG. 2 is a ribbon diagram illustrating the CocE structure. Domain 1(DOM1), domain 2 (DOM2), and domain 3 (DOM 3) are indicated, along withthe active site located at the intersection of the three domains. Abenzoic acid molecule is shown in the active site.

FIG. 3 is a line plot depicting the elution profile for CocE by FPLC(Q-Sepharose), where UV absorbance and sodium chloride concentration isshown over time. For further methodology information, see Example 1.

FIG. 4 is a line and scatter plot showing the in vitro degradation ofcocaine in the presence of CocE in human plasma. For further methodologyinformation, see Example 2.

FIG. 5 is Michaelis-Menten enzyme kinetics plot for wild-type CocE, theT172R mutant CocE (SEQ ID NO: 26), and the S159A mutant CocE (SEQ ID NO:11) for reactions carried out at 30° C. and 37° C. For furthermethodology information, see Example 4.

FIG. 6 is a set of photographs of the total protein staining after PAGEseparation of wild-type and T172R mutant CocE using denaturing (SDS+βMe)or non-denaturing (Native) conditions after incubation at 37 C forvarious time points. For more information see Example 4.

FIG. 7 is a line plot depicting the circular dichroism determinedmelting temperatures of wild-type and T172R CocE. The full spectrum isshown in A, the near-UV spectrum is shown in B, and the estimatedmelting temperature for each mutant is shown in C. For more informationsee Example 4.

FIG. 8 contains (A) a drawing of the hydrolysis of the benzoylthioesterderivative of cocaine (thio-1) and the des-carbomethoxy cocaine (thio-2)followed by reaction of the released thiols (R-SH) with Ellman'sreagent; and (B) a line plot showing the colorimetric reaction ofcontrol BL21 cells and BL21 cells containing CocE incubated with thebenzoylthioester derivative (thio-1) and Ellman's reagent. For moreinformation see Example 15.

FIG. 9 is a line and scatter showing the effect of 1.0 mg CocE (closedcircles) or PBS (vehicle, open circles) on cocaine-induced lethalitywhen administered one minute after increasing doses of cocaine (n=6-7).Data presented are expressed as percent and its standard error. Forfurther methodology information, see Example 5.

FIG. 10 is a bar graph showing the effect of increasing doses of CocE(CE) or human BChE (BChE), or PBS, on cocaine-induced lethality whenadministered one minute before 180 mg/kg cocaine (n=6-7). Data presentedare expressed as percent and its standard error mean. For furthermethodology information, see Example 5.

FIG. 11 is a bar graph showing the effect of CocE mutants S117A andY44F, or PMSF treated CocE on cocaine-induced convulsions and lethalitywhen administered 1 minute before 180 mg/kg cocaine (n=5-6). For furthermethodology information, see Example 5.

FIG. 12 is a bar graph showing the effect of 1.0 mg CocE or PBSadministered one minute after the 560 mg/kg WIN-35065-2, the determinedLD100 of the compound (n=6-8). For further methodology information, seeExample 5.

FIG. 13 is a line and scatter plot showing time-dependent inactivationof CocE (125 ng/ml) in vitro. Exponential decay t_(1/2) was calculatedas 13.2. For further methodology information, see Example 6.

FIG. 14 is a bar graph showing time-dependent protective effects of 1 mgCocE when administered at various times before and after 100 mg/kgcocaine (n=6-8) For further methodology information, see Example 6.

FIG. 15 is a bar graph showing the cocaine concentrations in humanplasma treated with 0.8 μM cocaine esterase or esterase vehicle at zeroand one minute following esterase administration. For furthermethodology information, see Example 6.

FIG. 16 is a series of line and scatter plots showing the effect of 0.32mg CocE (closed circles), 0.32 mg of T172R mutant CocE (closetriangles), or PBS (vehicle, open circles) on cocaine-induced lethalitywhen administered one minute after increasing doses of cocaine (leftpanel) and minutes to death after cocaine administration (right panel).For further methodology information, see Example 7.

FIG. 17 is a line and scatter plot showing the effect of 0.32 mg/kg ofCocE (closed circles) or T172R mutant CocE (closed triangles) oncocaine-induced lethality when administered at 1, 10, 30, and 60 minutesbefore administration of 320 mg/kg cocaine (n=6). For furthermethodology information, see Example 7.

FIG. 18 is a series of line and scatter plots showing the effect of 1 mgof hBChE (closed triangle), 0.32 mg CocE (closed circle), or 1 mg ofCocE (closed square) on cocaine-induced lethality. FIG. 18A showslethality as a function of cocaine concentration. FIG. 18B shows minutesuntil death after cocaine administration as a function of cocaineconcentration. For further methodology information, see Example 8.

FIG. 19 is a trace of the MALDI-TOF mass spectrum of pegylated CocE.Mass difference between peaks corresponds to ˜5500 Da; equivalent to themolecular weight of a single PEG chain. For further methodologyinformation, see Example 11.

FIG. 20 is an SEM image of gluteraldehyde fixed red blood cells. FIG.20A is normal RBC without treatment. FIG. 20B is osmotic ruptire-resealRBC loaded with L-ASNase. FIG. 20C is RBC loaded with LMWP-ASNase. Forfurther methodology information, see Example 12.

FIG. 21 is a scatter plot showing percent Asperaginase activity in bloodas a function of time (days) for RBC/LMP-ASNase or RBC ghost/ASNase. Forfurther methodology information, see Example 12.

FIG. 22 is a line plot showing survival of DBA/2 mice bearing L5178Ylymphoma cells. Enzyme or saline were given on day 5, at which timesymptoms were present. For further methodology information, see Example12.

FIG. 23 is a line and scatter plots demonstrating protective effects ofCocE and its mutants against cocaine-induced toxicity. The figuredepicts percentage of mice lethality exhibiting cocaine-inducedlethality as a function of injected cocaine concentration in miceadminstered wild type CocE (0.3 mg), T172R (0.3 mg), T172R-G173Q (0.3mg), or L169K (1 mg). CocE or mutants (mg) was administeredintravenously 1 minute before cocaine administration (mg/kg, i.p.).Different symbols represent dose-response curves of cocaine-inducedlethality in the absence or presence of CocE or mutants. Each data pointrepresents the percentage of mice (n=8 for each dosing condition)exhibiting cocaine-induced lethality.

FIG. 24 is a series of line and scatter plots demonstrating time courseof protective effects of CocE against cocaine toxicity. CocE or mutants(0.1, 0.3, and 1 mg, i.v.) was administered at different time pointsbefore administration of i.p. cocaine 180 mg/kg. FIG. 24A depictspercentage of mice exhibiting cocaine-induced lethality as a function oftime of administration of wild type CocE (0.1 mg), T172R (0.1 mg), L169K(0.1 mg), or T172R-G173Q (0.1 mg) before administration of i.p. cocaine180 mg/kg. FIG. 24B depicts percentage of mice exhibitingcocaine-induced lethality as a function of time of administration ofwild type CocE (0.3 mg), T172R (0.3 mg), L169K (0.3 mg), or T172R-G173Q(0.1 mg) before administration of i.p. cocaine 180 mg/kg. FIG. 24Cdepicts percentage of mice exhibiting cocaine-induced lethality as afunction of time of administration of wild type CocE (1 mg), T172R (1mg), L169K (1 mg), or T172R-G173Q (1 mg) before administration of i.p.cocaine 180 mg/kg. Each data point represents the percentage of mice(n=8 for each dosing condition) exhibiting cocaine-induced lethality.

FIG. 25 is a line and scatter plot demonstrating estimated duration ofprotection for 50% lethality. The figure depicts the estimated duration(hours) of protection (50% lethality) of CocE mutants in mice as afunction of dosage (mg, i.v.) of T172R-G173Q, L169K, T172R, and wildtype CocE. The time required to reach 50% lethality was determined fromFIG. 24.

FIG. 26 is a series of line and scatter plots demonstrating protectiveeffects of CocE, T172R-G173Q, and their pegylated forms againstcocaine-induced toxicity. Each enzyme (0.3 mg) was administeredintravenously 1 min before cocaine administration (mg/kg, i.p.).Different symbols represent dose-response curves of cocaine-inducedlethality in the absence or presence of the enzyme. Each data pointrepresents the percentage of mice (n=8 for each dosing condition)exhibiting cocaine-induced lethality. FIG. 26A depicts the percentageoccurrence of lethality as a function of cocaine dosage (mg/kg, i.p.)for Vehicle/PBS, CocE wild type (0.3. mg), and PEG-CocE wild type (0.3mg). FIG. 26B depicts the percentage occurrence of lethality as afunction of cocaine dosage (mg/kg, i.p.) for Vehicle/PBS, T172R-G173Q(0.3. mg), and PEG-T172R-G173Q (0.3. mg).

FIG. 27 is a series of line and scatter plots showing the thermalstability of cocaine esterase and mutants: (A) wild-type CocE, (B)T172R, (C) T272R/G173Q and L169K. For further methodology information,see Example 17.

FIG. 28 is a series of chromatograms showing enzymes preincubated at 37°C. for 0 minutes or 60 minutes and resolved by size exclusionchromatography (A) wild-type CocE, (B) T172R, (C) T172R/G173Q. Molecularweight standards, BSA (66 Kda) and AD (150 Kda) are included in A. Forfurther methodology information, see Example 18.

FIG. 29 is a series of smoothed CD spectra of wild-type CocE and fourmutants showing temperature dependent melting observed between 200 and250 nm. For further methodology information, see Example 19.

FIG. 30 is a series of spectra for wild-type CocE and four mutantsdeconvoluted to 3 curves via the CCA algorithm suggesting that themelting of CocE is at least a two-step process moving from he originalcurve (curve 1) to an intermediate unfolding step (curve 2) to a fullydenatured protein (curve 3). For further methodology information, seeExample 19.

FIG. 31 is a series of line and scatter plots showing percentagecontribution of each temperature in describing the 3 deconvoluted CCAcurves shown in FIG. 30. For further methodology information, seeExample 19.

FIG. 32 is a line and scatter plot showing melting and formationtemperatures for (1) the initial spectra melts, (2) the formation andmelt of the intermediate state, and (3) the accumulation of the fullymelted protein. For further methodology information, see Example 19.

FIG. 33 is a picture of a gel showing that Cocaine (mM range prevented37° C.-induced formation of high molecular weight CocE aggregates (0.1mg/ml enzyme concentrations. For further methodology information, seeExample 20.

FIG. 34 is a line and scatter plot showing that Cocaine (uM quantities)stabilized 37° C.-induced activity loss. For further methodologyinformation, see Example 20.

FIG. 35 is a picture of a gel showing that Benzoic acid (mM rangeprevented 37° C.-induced formation of high molecular weight CocEaggregates (0.1 mg/ml enzyme concentrations. For further methodologyinformation, see Example 20.

FIG. 36 is a line and scatter plot showing that Cocaine (uM quantities)stabilized 37° C.-induced activity loss. For further methodologyinformation, see Example 20.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention disclosed wherein provide compositions andmethods for a catalytic degradation approach to anti-cocainetherapeutics. The technology described herein is based in part on thediscovery of highly efficient, thermostable, and long-lasting cocaineesterase mutants that can protect against the toxic and reinforcingeffects of cocaine in subjects. Such mutants provide treatment optionsfor cocaine-induced conditions such as cocaine overdose and cocaineaddiction.

Mutant CocE Polypeptides

Despite the potency of wild-type CocE (see e.g., SEQ ID NO: 1, AccessionNo. AF173165) in metabolizing cocaine (see e.g., Example 2; Example 4;Example 5), application of wild-type CocE as a therapeutic agent in thetreatment of cocaine overdose may be limited because of its low thermalstability at the physiological temperature (se e.g., Example 4; Example6). Thermo-instability contributes to the short plasma half-life ofwild-type CocE. Significant decay (>50%) of CocE activity was observedfollowing incubation of the enzyme in plasma at 37° C., or after itsintravenous administration into a mouse. The t_(1/2) of CocE at 37° C.is approximately 15 minutes whereas at 4° C. the t_(1/2) is greater than6 months. Preliminary studies in rats demonstrated a relatively shortduration of anti-cocaine effect of little more than 30 minutes for CocE.

One aspect of the invention thus provides purified mutant CocEpolypeptides that exhibit increased thermal stability and plasmahalf-life as compared to wild-type CocE. The mutant CocE polypeptides ofthe invention hold significant clinical value because of theircapability to efficiently hydrolyze cocaine, while also exhibitingincreased thermostability and/or plasma half-life.

The invention provides mutant CocE polypeptides in which at least oneamino acid residue of the wild-type CocE is substituted, where themutant CocE has increased thermostability while retaining relativelyhigh catalytic efficiency. In some embodiments, mutant CocE polypeptidessubstantially maintain the wild-type CocE polypeptide functionalesterase activity (i.e., hydrolysis of cocaine). Mutant CocEpolypeptides have a peptide sequence that differs from a native CocEpolypeptide in one or more amino acids. The peptide sequence of suchmutants can feature a substitution, deletion, or addition of one or moreamino acids of a native CocE polypeptide. Amino acid insertions arepreferably of about 1, 2, 3, and 4 to 5 contiguous amino acids, anddeletions are preferably of about 1, 2, 3, 4, 5, 6, 7, 8, and 9 to 10contiguous amino acids. In various embodiments, the mutant CocEpolypeptide can contain at least one, two, three, four, or more aminoacid substitutions, deletions, or additions, where the resulting mutantCocE polypeptide has increased thermostability.

The term amino acid, as used herein, is intended to include naturallyoccurring amino acids as well as non-naturally occurring amino acids,including amino acid analogs and derivatives. The latter includesmolecules containing an amino acid moiety. One skilled in the art willrecognize that reference herein to an amino acid includes, for example,naturally occurring proteogenic L-amino acids; D-amino acids; chemicallymodified amino acids such as amino acid analogs and derivatives;naturally occurring non-proteogenic amino acids, and chemicallysynthesized compounds having properties known in the art to becharacteristic of amino acids. For all the amino acid sequencesdisclosed herein, it is understood that equivalent nucleotides and aminoacids can be substituted into the sequences without affecting thefunction of the sequences. Such substitution is within the ability of aperson of ordinary skill in the art.

The invention also provides purified mutant CocE polypeptides with thefollowing substitutions: L163V (SEQ ID NO: 3); V121D (SEQ ID NO: 4);S167A (SEQ ID NO: 5); Q123E (SEQ ID NO: 6); V225I (SEQ ID NO: 7); I218L(SEQ ID NO: 8); A310D (SEQ ID NO: 9); A149S (SEQ ID NO: 10); S159A (SEQID NO: 11); S265A (SEQ ID NO: 12); S56G (SEQ ID NO: 13); W220A (SEQ IDNO: 14); T122A (SEQ ID NO: 15); S140A (SEQ ID NO: 16); F189L (SEQ ID NO:17); A193D (SEQ ID NO: 18); T254R (SEQ ID NO: 19); N42V (SEQ ID NO: 20);V262L (SEQ ID NO: 21); L508G (SEQ ID NO: 22); Y152H (SEQ ID NO: 23);V160A (SEQ ID NO: 24); T172R (SEQ ID NO: 25); Y532F (SEQ ID NO: 26);T74S (SEQ ID NO: 27); W285T (SEQ ID NO: 28); L146P (SEQ ID NO: 29);D533S (SEQ ID NO: 30); A194R (SEQ ID NO: 31); G173Q (SEQ ID NO: 32);C477T (SEQ ID NO: 33); K531A (SEQ ID NO: 34); R41I (SEQ ID NO: 35);L119A (SEQ ID NO: 36); K46A (SEQ ID NO: 37); F84Y(SEQ ID NO: 38),T172R-G173Q (SEQ ID NO: 39); L169K (SEQ ID NO: 40); F189A (SEQ ID NO:41), N197K (SEQ ID NO: 42), R182K (SEQ ID NO: 43), F189K (SEQ ID NO:44), V190K (SEQ ID NO: 45), Q191K (SEQ ID NO: 46), and A194K (SEQ ID NO:47). For example, the T172R mutant CocE polypeptide (SEQ ID NO: 25) hasincreased thermostability, increased Vmax and Km at 37° C., increasedmelting temperature (Tm), increased plasma half-life, greater reductionsin lethality due to cocaine toxicity, and longer lasting anti-cocaineeffects, as compared to wild-type CocE (see e.g., Example 4, Example 7).

The resulting increase in thermostability of the mutant CocE polypeptideis at least about 2 kcal/mol. Thermostability of a given polypeptide canbe assessed by a variety of methods known to the art, including forexample circular dichroism (CD) spectroscopy or differential scanningcalorimeter. For example, the resulting increase in thermostability canbe at least about 2.1, at least about 2.2, at least about 2.3, at leastabout 2.4, at least about 2.5, at least about 2.6, at least about 2.7,at least about 2.8, at least about 2.9, at least about 3.0, at leastabout 3.1, at least about 3.2, at least about 3.3, at least about 3.4,at least about 3.5, at least about 3.6, at least about 3.7, at leastabout 3.8, at least about 3.9, at least about 4.0, at least about 4.1,at least about 4.2, at least about 4.3, at least about 4.4, or at leastabout 4.5 kcal/mol. Even greater thermostability increases arecontemplated. It is thought that lowering the energy by about 2.1 toabout 4.5 kcal/mol can extend the half-life time of the protein about 30to about 1000 fold longer at room temperature.

Generally, the mutant CocE polypeptides have esterase activity withincreased thermostability as compared to wild-type CocE. In someembodiments, the thermostable mutant CocE polypeptides can have lessesterase activity than wild-type CocE. For example, thermostable CocEmutants can have about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 99% of the esterase activity of wild-type CocE.In other embodiments, the mutant CocE polypeptides have approximatelythe same, or greater, catalytic efficiency of wild-type CocEpolypeptides. For example, thermostable CocE mutants can have about100%, about 110%, about 120%, about 130%, about 140%, about 150%, ormore of the esterase activity of wild-type CocE.

Variants of the mutant CocE polypeptides such as fragments, analogs, andderivatives are also within the invention. CocE polypeptide fragmentscorresponding to one or more particular motifs and/or domains or toarbitrary sizes, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 75, 100, 125, 150, 200, 250,300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1150 and 1200 amino acidsin length are intended to be within the scope of the invention disclosedherein. Isolated peptidyl portions of CocE polypeptides can be obtainedby screening peptides recombinantly produced from the correspondingfragment of the nucleic acid encoding such peptides. In addition,fragments can be chemically synthesized using techniques known in theart such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. For example, a CocE polypeptide as described herein can bearbitrarily divided into fragments of desired length with no overlap ofthe fragments, or preferably divided into overlapping fragments of adesired length.

Another aspect of the invention disclosed herein concerns recombinantforms of the CocE polypeptide. In some embodiments, isolated nucleicacid molecules of the invention include those polynucleotides encodingthe above described CocE polypeptides. In other embodiments, therecombinant polypeptides of the invention disclosed herein are encodedby a nucleic acid that has at least 85% sequence identity (e.g., 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and99%) with the nucleic acid sequence of SEQ ID NO: 2, where the expressedrecombinant CocE polypeptide retains substantially the same or morecatalytic efficiency of wild-type CocE polypeptide and has increasedthermostability as compared to wild-type CocE.

Nucleic acids that hybridize under stringent conditions to the nucleicacids of SEQ ID NO: 2 or the complements of SEQ ID NO: 2 can also beused in the invention. For example, such nucleic acids that hybridize toSEQ ID NO: 2 or the complement of SEQ ID NO: 2 under low stringencyconditions, moderate stringency conditions, or high stringencyconditions and also encode a mutant CocE polypeptide that has esteraseactivity with increased thermostability as compared to wild-type CocE,are within the invention. Preferred nucleic acids are those having anucleotide sequence that is the complement of all or a portion of SEQ IDNO: 2. Other variants of the native CocE gene within the invention arepolynucleotides that share at least 65% (e.g., 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, and 99%) sequence identity to SEQ ID NO: 2 or the complement of SEQID NO: 2. Nucleic acids that hybridize under stringent conditions to orshare at least 65% sequence identity with SEQ ID NO: 2 or the complementof SEQ ID NO: 2 can be obtained by techniques known in the art such asby making mutations in the native CocE gene, or by isolation from anorganism expressing such a nucleic acid (e.g., an allelic variant).

Nucleic acid molecules encoding mutant CocE fusion proteins are alsowithin the invention. Such nucleic acids can be made by preparing aconstruct (e.g., an expression vector) that expresses mutant CocE fusionprotein when introduced into a suitable host. For example, such aconstruct can be made by ligating a first polynucleotide encoding mutantCocE protein fused in frame with a second polynucleotide encodinganother protein such that expression of the construct in a suitableexpression system yields a fusion protein.

The nucleic acid molecules of the invention can be modified at a basemoiety, sugar moiety, or the phosphate backbone, e.g., to improvestability of the molecule, hybridization, and the like. Nucleic acidmolecules utilized in embodiments of the invention disclosed herein canbe in the form of RNA or in the form of DNA (e.g., cDNA, genomic DNA,and synthetic DNA). The DNA can be double-stranded or single-stranded,and if single-stranded can be the coding (sense) strand or non-coding(anti-sense) strand. The coding sequence which encodes a mutant CocEpolypeptide can be identical to the claimed nucleotide sequence, or itcan also be a different coding sequence which, as a result of theredundancy or degeneracy of the genetic code, encodes the samepolypeptide as the polynucleotides of SEQ ID NOS: 3-37.

Design of Mutant CocE

CocE thermostabilizing mutations can be designed to either increase thethermodynamic stability of a polypeptide through loweringΔG(unfolded→folded) or decrease rate of unfolding through increasing theactivation free energy of the unfolding process. The ΔG(unfolded→folded)is the free-energy difference between the unfolded and the folded state.For a thermodynamically stable polypeptide, ΔG(unfolded→folded) shouldbe a negative value. Generally, the lower the ΔG(unfolded→folded) value,the more stable the folded state. The activation free energy of theunfolding process is the free energy difference between the folded stateand the transition state of unfolding (Steipe, 1999).

Mutant CocE polypeptides with increased thermostability can be designedand generated by a variety of methods known to the art including, forexample, rational design, directed evolution (e.g., random mutagenesis,mutagenesis of the CocE host organism), or a combination thereof.Directed evolution can be achieved through mutation and recombinationfollowed by either screening for the desired trait or by applying aselective pressure to obtain the trait of interest (see e.g., Lehmannand Wyss, 2001). Mutagenesis can be performed either on the specificgene of interest or through mutagenesis and selection of a host organismsuch that the engineered property is conferred to the protein ofinterest. Preferably, the thermostability of CocE mutants are engineeredthrough a three-prong approach of (i) rational design throughcomputation-guided site-directed mutagenesis (see e.g., Example 3;Example 4), (ii) random mutagenesis of the CocE gene and high throughputscreening (see e.g., Example 4; Example 15); and (iii) mutagenesis ofthe CocE host organism followed by genetic selection (see e.g., Example14).

Various rational design concepts are known to those of skill in the artto accomplish lowering ΔG(unfolded→folded) and/or increasing theactivation free energy of the unfolding process (see e.g., Lehmann,2001). For example, one can: decrease the entropy of the unfolded stateby introducing additional disulfide bridges or by X→Pro mutations;increase a-helix propensity by Gly→Ala substitutions or by stabilizationof a-helix macrodipoles; improve electrostatic interactions betweencharged surface residues by introducing additional salt bridges or evensalt-bridge networks, or by thermostabilizing mutations based oncalculations of electrostatic potentials.

Molecular modeling, based on appropriate molecular dynamics (MD)simulations, can rationally guide site-directed mutagenesis so as todesign mutant CocE polypeptides possessed of increased thermostability.Classical MD simulation enables the study of time evolution of a largesystem by taking many small successive time steps under atomic forcesdetermined by a set of parameterized interaction functions (forcefield), including bonded interactions (bonds, angles, and dihedralangles), non-bonded van der Waals interactions, and electrostaticinteractions based or net atomic charges. Due to the simple force fieldform, the MD simulation may be performed for a sufficiently longsimulation time to give meaningful ensemble-averaged properties, evenfor a very large system involving over a hundred thousand atoms. So, forCocE and each proposed mutant, the MD simulation can lead to areasonable, dynamically averaged 3D structure of the simulatedpolypeptide in water.

One approach used successfully herein focuses on the rational design ofthermostabilizing mutations that lower the ΔG(unfolded→folded) value ofthe polypeptide (see e.g., Example 3). Such approach requires only thecalculation of ΔG(unfolded→folded), without performing a moretime-consuming computation on the structure and energetics of thetransition state of the unfolding. Hence, to increase thethermostability of a polypeptide before pegylation, one can use a methodimplemented in a rational design program (e.g., RosettaDesign) that usesan energy function for evaluating the fitness of a particular sequencefor a given fold and a Monte Carlo search algorithm for samplingsequence space. Such approach is known to produce increasedthermostability of other enzymes with no reduction in catalyticefficiency (see e.g., Korkegian, 2005). For example, the fold used inthe computation can correspond to that of the available CocE X-raycrystal structure.

The rational design program allows prediction of a set of modified aminoacid sequences that potentially have lower energies (e.g., theΔG(unfolded→folded) values) and, therefore, higher thermostability.Thus, one can use the computational design described herein to predictmutations in the CocE polypeptide core that can lead tothermostabilization of the polypeptide without loss of catalyticefficiency. This approach minimizes experimental test time and greatlyincreases the success of experimental outcomes. The predictedthermostabilizing mutations can be tested individually by site-directedmutagenesis and then in combination, in an iterative process (see e.g.,Example 15).

Directed evolution can also be used to generate thermostable CocEmutants. Directed evolution encompasses a series of experimentaltechniques that produce accelerated diversity and adaptation throughmutation and recombination followed by either screening for the desiredtrait or by applying a selective pressure to obtain the trait ofinterest (Lehmann & Wyss (2001) Current Opinion in Biotechnology 12,371-375). Thus directed evolution involves both a process to generatediversity and an efficient screening or selection method for thedetection or enrichment of the desired trait. Directed evolution haspreviously been successfully applied to the production of thermostableproteins, and the generation of diversity has been achieved through, forexample, error prone PCR, saturation mutagenesis, DNA shuffling,chemical mutagenesis, and combinations thereof. Error prone PCRamplifies the gene of interest with non-proofreading polymerases andstressful conditions designed to randomly generate single base pairmutations. After each round the best mutants are selected and are usedas parent sequences in the next round of mutagenesis. This technique hasbeen used to generate a number of thermostable protein variants,including propyl endopeptidase (Uchiyama ma., et al. 2000),betaglucuronidase (Flores, H. and A. D. Ellington (2002) Journal ofMolecular Biology 315, 325-337) and family 10 xylanases (Andrews et al.(2004) J Biol Chem 279, 54369-79). Saturation mutagenesis also amplifiesthe gene of interest but incorporates universal bases duringamplification to generate a much higher number of mutations. Thistechnique has been used to generate thermostability in a psychrophilicenzyme (Miyazaki et al. (2000) Journal of Molecular Biology 297,1015-1026). DNA shuffling involves one or more cycles of recombinationbetween a set of homologous sequences to obtain improved variants of agiven enzyme. This technique can also be used in tandem with error-pronePCR, where the best mutants obtained by error-prone PCR are combined byDNA shuffling to generate a new subset of mutants. DNA shuffling hasbeen implemented to generate thermostable variants of beta-glucuronidase(Flores, H. and A. D. Ellington (2002) Journal of Molecular Biology 315,325-337). Chemical mutagenesis involves treating plasmid DNA withchemicals that introduce point mutations into the sequence such ashydroxylamine, nitrosamines or dimethyl sulfate. Plasmid treatment withhydroxylamine has been used to generate thermostable mutants of Bacilluspolymyxa beta-glucosidase A (Lopez-Camacho et al. (1996) BiochemistryJournal 314, 833-838) and firefly luciferase (White et al. (1996)Biochemistry Journal 319 (Pt 2), 343-350).

Mutagenesis of the CocE host organism can also be used to generatethermostable CocE mutants. A simple and fast method for the productionof thermostable CocE variants can be achieved by utilizing the enzyme'sability to confer metabolism of cocaine as a sole carbon source on itshost organism. The CocE gene was originally sequenced from RhodococcusMB1 by subcloning gene fragments into Rhodococcus erythropolis CW25, abacterium unable to metabolize cocaine but able to grow on the cocaineesterase byproducts ecgonine methyl ester and benzoate (Bresler et al.(2000) Applied & Environmental Microbiology 66, 904-908). Anotherorganism previously shown to metabolize cocaine byproducts isPseudomonas fluorescens (MBER), which was able to grow in a symbioticrelationship with another bacterium able to metabolize cocaine via theesterase Comamonas acidovorans (MBLF).

While difficult to transform with plasmids at high efficiencies, thesebacterial strains can be relatively easily transformed with the nativeCocE gene cloned into appropriate shuttle vectors and then traditionalbacterial mutagenesis can be performed (see e.g., Example 14). Becausethese bacteria would normally only grow at 25-30° C. on cocainehydrolysis products, selection for mutants able to metabolize cocaineefficiently at 37° C. would be expected to select for highly active andCocE mutants that are stable at 37° C.

The exposure of bacteria to radiation or chemical agents for theproduction of mutants carrying new phenotypes is well known in the art(see e.g., Maron, D. M. and Ames, B N (1983) Mutation Research 113,173-215). Mutagenesis by irradiation can involve both ionizing andnon-ionizing radiation; however, non-ionization radiation is the widelyused, and UV radiation at 260 nm is most effective as a lethal agent.Mutagenesis is caused by the induction of pyrimidine dimers, increasingthe likelihood of incorporating mismatches during replication. Cells areexposed to UV radiation at a dose predetermined to kill 90-95% of thecell population, and mutants are then sought among the survivors.Chemical mutagenesis includes the use of base analogs such as5-bromouracil and 2-aminopurine which increase copy error duringreplication, or the use of agents that react directly with DNA such ashydroxylamine or nitrosoguanidine which induce mutations at a higherfrequency than base analogs.

Generating Mutant CocE Polypeptides

Embodiments of the invention further pertain to methods of producing themutant CocE polypeptides. For example, a host cell transfected with anucleic acid vector directing expression of a nucleotide sequenceencoding the mutant CocE polypeptides can be cultured under appropriateconditions to allow expression of the peptide to occur. The cells can beharvested, lysed, and the protein isolated. A mutant CocE polypeptidecan be isolated from host cells using techniques known in the art forpurifying proteins including ion-exchange chromatography, gel filtrationchromatography, ultrafiltration, electrophoresis, and immunoaffinitypurification with antibodies specific for such protein (see e.g.,Example 1).

For example, after a mutant CocE polypeptide has been expressed in acell, it can be isolated using any immuno-affinity chromatography. Morespecifically, an anti-CocE antibody can be immobilized on a columnchromatography matrix, and the matrix can be used for immuno-affinitychromatography to purify the mutant CocE polypeptide from cell lysatesby standard methods (see, e.g., Sambrook and Russel (2006) CondensedProtocols from Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, ISBN 0879697717). After immuno-affinitychromatography, the mutant CocE polypeptide can be further purified byother standard techniques, e.g., high performance liquid chromatography.In another embodiment, a mutant CocE polypeptide is expressed as afusion protein containing an affinity tag (e.g., His×6) that facilitatesits purification (see e.g. Example 1).

Pegylation of Mutant CocE Polypeptides

The mutant CocE can be pegylated so as to increase the duration ofaction and heat stability, and decrease immunogenicity. Pegylation canfurther enhance the thermostability of the mutant CocE of the inventionand increasing serum half life by decreasing renal clearance,proteolysis, macrophage uptake, and immunological response.

Pegylation is the process of attaching repeating units of ethyleneglycol (i.e., polyethylene glycol, or PEG) to a polypeptide to reducethe polypeptide's immunogenicity and its rate of renal clearance (seegenerally (Veronese, F M and Harris, J M (2002b) Advanced Drug DeliveryReviews 54, 457-606, Veronese, F M and Harris, J M (2002c) Advanced DrugDelivery Reviews 55, 1259-1345, reviewing PEGylation technology). Eachethylene glycol unit can bind to two or three water molecules, whicheffectively increases the size of the peptide, and can protect thepeptide from immune responses, enzymatic degradation, and/or rapid renalclearance. The polyethylene glycol can also stabilize against changes intemperature and pH. The net result is that the therapeutic polypeptidecan be maintained longer in the blood, and induces a lesser immuneresponse (Harris, J M and Chess, R B (2003) Nature Reviews. DrugDiscovery 2, 214-221). PEG possesses a unique set of properties,including absence of toxicity, antigenicity, and immunogenicity, amass-dependent diminution of renal clearance, and a high flexibility andsolubility in water. It imparts these characteristics to the proteins towhich it is bound (Veronese, F M and Harris, J M (2002b) Advanced DrugDelivery Reviews 54, 457-606).

A PEG polymer can first be activated with a functional group thatencourages covalent binding to an amino acid of the protein. Theterminal hydroxyl group of the PEG can be modified by an activecarbonate, active ester, aldehyde or tresylate derivative. The PEG canbe attached to lysines or to introduced cysteine residues of the mutantCocE. Repeating units of ethylene oxide can be constructed in manyconfigurations having different lengths, with or without branching, andwith various molecular weights. Means of incorporation can includesite-directed mutagensis or use of maleimide derivatives oftransglutaminase.

PEG is FDA approved for use as a vehicle or base in pharmaceuticals,including injectable, topical, rectal and nasal formulations (Harris andChess, 2003). And PEGylated drugs have been approved for clinical use(see e.g., PEG-interferon alpha-2a (Hamidi, M and Tajerzadeh, H (2003)Drug Delivery 10, 9-20; PEG-interferon alpha-2b (Reddy et al. (2002)Advanced Drug Delivery Reviews 54, 571-586).

Owing to the unparallel spatial dynamic mobility of PEG, modification ofthe surface of a mutant CocE by the PEG polymer can shield the coatedenzyme from actions by the proteolytic enzymes and recognition by theimmune system; thereby reducing the immunogenicity and prolonging thecirculating half-life of the pegylated mutant CocE. Success ofpegylation strategies include PEG-modified liposomes (i.e., stealthliposomes, so termed due to their ability in dodging immune detectionand renal clearance to yield a significantly extended circulating time(Lasic, DD (1997) Journal of Controlled Release 48, 203-222), as well aspegylated asparaginase whose in vivo half-life has been drasticallyimproved from 26 hours seen for free asparaginase to 15 days (Avramis etal. (2002) Blood 99, 1986-1994). Further, it is recognized in the artthat pegylation can significantly enhance the thermal stability of anenzyme (Kazan, D. and Erarslan, A. (1997) Applied Biochemistry &Biotechnology 62, 1-13; Efremova et al. (1998) Biochemistry (Moscow) 63,441-447) and reduce activation of the complement system (Chang et al.(2005) Bioconjugate Chemistry 16, 147-155). Thus, PEGylation technologyis well suited to improve the pharmacological and pharmaceutical effectsof the mutant CocE of the invention (see e.g., Example 11). Pegylationof mutant CocE can also be used in conjunction with RBC encapsulation.

Encapsulating Mutant CocE in RBC

The mutant CocE can be encapsulated into red blood cells (RBC) so as toincrease the duration of action and heat stability, and decreaseimmunogenicity. Cocaine, just as it rapidly crosses the blood-brainbarrier, also rapidly transits the RBC plasma membrane, and this uptakehas been demonstrated in a study of intravenous cocaine administrationin man (Javaid et al. (1978) Journal of Chromatography 15, 105-113).Furthermore, cocaine concentrations in RBC exceed the concentrations inplasma (Javaid et al. (1978) Journal of Chromatography 15, 105-113). Assuch, RBC encapsulation can be utilized to shield thermostable forms ofCocE from clearance.

Erythrocytes have been exploited extensively for their potentialapplications as drug carriers (Wang et al. (2002) Advanced Drug DeliveryReviews 54, 547-570). Being the most abundant cells of the human body,RBC offers unmatched advantages to function as a drug carrier. First,erythrocytes are completely biocompatible and biodegradable,particularly when the autologous cells are used for drug loading. Inaddition, the biconcave disk shape of erythrocytes provides them withthe highest surface to volume ratio (1.9×10⁴ cm/g) (Guyton, A G & Hall,J E (1996) Textbook of Medical Physiology, 425-433) usable for drugencapsulation. Encapsulation in RBC also prevents the loaded drug frominactivation by endogenous factors as well as protects the organismsfrom toxic effects of the encapsulated drug (Wang et al. (2002) AdvancedDrug Delivery Reviews 54, 547-570). Furthermore, RBC encapsulationenables avoidance of undesirable immune responses (i.e. antigenicity andimmunogenicity) that may be triggered by foreign bodies (Wang et al.(2002) Advanced Drug Delivery Reviews 54, 547-570). And, erythrocytespossess longer circulation half-life in comparison to other syntheticcarriers. For example, the normal life-span of an erythrocyte insystemic circulation is reported to be about 120 days (Guyton, A G &Hall, J E (1996) Textbook of Medical Physiology, 425-433).

Encapsulation of mutant CocE in erythrocytes can be according to severaltechniques known in the art, including electroporation, drug (e.g.,primaquine)-induced endocytosis, and osmosis (see e.g., Green, R. andWidder, K J (1987) Methods In Enzymology, Vol. 149). These methods caninclude swelling and disrupting the cell membrane, releasing theinterior content including primarily hemoglobin and also cytoskeleton,entrapping the drug, and then resealing the membrane to yield thecup-shaped, spherical, pink or white-colored RBC (generally termed“RBC-Ghost”).

Alternatively, membrane-penetrating peptides can be employed toencapsulate mutant CocE in erythrocytes (see e.g., Example 12).Membrane-penetrating peptides, or protein transduction domain (PTD)peptides are a family of small (normally consists of 10-15 amino acidresidues) peptides including TAT, LMWP, and other arginine-rich cationicpeptides (see generally, Dietz, G P H and Bahr, M (2004) Molecular CellNeurosciences 27, 85-131). It is known to the art that, by covalentlylinking PTD to almost any type of molecular species including proteins(MW>150 kDa; more than 60 different proteins have already been tested(Dietz, G P H and Bahr, M (2004) Molecular Cell Neurosciences 27,85-131) and nano-carriers (e.g. liposomes), PTD can ferry the attachedspecies across cell membrane of all organ types including the brain(Schwarze et al. (1999) Science 285, 1569-1572). PTD is neither toxicnor immunogenic (Schwarze et al. (1999) Science 285, 1569-1572), and thePTD-mediated cell internalization does not induce perturbation oralteration of the erythrocytes cell membrane (Suzuki et al. (2002)Journal of Biological Chemistry 25, 2437-2443). PTD has been usedsuccessfully in protein loading RBC, and results in erythrocytes withunaltered physical and chemical attributes (see e.g., Li et al. (2003)American Pharmaceutical Review 6, 22-26). Thus, PTD peptide conjugationto mutant CocE can facilitate RBC encapsulation. RBC encapsulation canalso be carried out by using pegylated mutant CocE.

Stabilizing Mutant CocE

Another aspect of the invention is directed toward stabilization ofmutant CocE polypeptides using substrates, products, and/or inhibitorsof cocaine. Substrates and products useful in embodiments disclosedherein include, for example, but are not limited to, cocaine; cocainederivatives, such as, for example, (−)-cocaine, (+)-cocaine,tropococaine, and the like; thio-cocaine derivatives, such as, forexample, Thiol-1, Thiol-2, and the like; amide-cocaine derivatives;provitamin-cocaine derivatives, such as PABA cocaine, Niacin cocaine,and the like; benzoic acid; 4-nitrophenyl acetate (4NPA); 4-nitrophenol(4NP); and the like. Exemplary inhibitors include, but are not limitedto, substrate analogues, such as, phospho-fluorococaine,O-Phospho-cocaine, O-methylphosphococaine, S-Methylphophococaine, andthe like; product analogues, such as, Ecgonine and Ecgonine derivatives,such as, boronic acid ecogonine methylester analog; phenylboronic acid;benzoic acid derivatives, such as, 4-tert-Butyl benzoic acid,1-Naphthoic acid, 2,3,4-trimethyl-benzoic acid methyl ester, and thelike. Additional chemicals include, for example, but are not limited to,SDS, glycerol, PEG, and the like.

Preferably, the substrates, products, and/or inhibitors stabilizethermal denaturation of the polypeptides disclosed herein. In someembodiments, the substrates, products and/or inhibitors also preventthermally-induced aggregation in gel electrophoresis. Generally, use ofa substrate, product, and/or inhibitor results in at least about a 10%increase in stability and/or inhibition, respectively. For example theincrease can be about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 150%, 200%, 300%, or even500% or greater. Thus, substrates, products, and/or inhibitors are wellsuited to stabilize the mutant CocE polypeptides disclosed herein (seee.g., Example 20).

In one embodiment, small molecules are used to thermostabilize themutant CocE polypeptides disclosed herein. In a preferred embodiment,such molecules do not occupy the active site of the polypeptide.

In some embodiments, the polypeptides disclosed herein can be co-infusedwith a stabilizing molecule. In other embodiments, the stabilizingmolecules can be used to stabilize the polypeptide during manufacturing.In still other embodiments, the stabilizing molecules can be used tostabilize the mutant CocE polypeptides until ready for use.

Treatment Methods

Another aspect of the invention is directed toward catalytic degradationapproach to anti-cocaine therapeutics. Provided are treatments, bothprophylactic and therapeutic, of cocaine-induced conditions through theadministration of thermostable, esterase active, mutant CocEpolypeptides to a subject in need thereof. The cocaine esterase variantsof the invention hold significant clinical value because of theirincreased thermostability and longer plasma half-life than knownnaturally occurring CocE. It is this increase in thermostability andplasma half-life that enables a much more rapid response to thelife-threatening symptoms of cocaine toxicity that sets the CocEvariants of the invention apart from other treatment options.

A determination of the need for treatment will typically be assessed bya history and physical exam consistent with the cocaine-inducedcondition. Cocaine-induced conditions include, but are not limited to,cocaine overdose, cocaine toxicity, and cocaine dependence and/oraddiction. For example, the diagnosis of cocaine toxicity can includeconvulsions, grand-mal seizures, cardiac arrest, myocardial infarction,cardiac arrhythmias, increased blood pressure, stroke, drug-inducedpsychosis, dissecting aneurysm, and increased myocardial oxygen demand.As another example, in the case of cocaine dependence and/or addiction,withdrawal symptoms include subjective sensations of mild to severedysphora, depression, anxiety, or irritability. Subjects with anidentified need of therapy include those with a diagnosedcocaine-induced condition, an indication of a cocaine-induced condition,and subjects who have been treated, are being treated, or will betreated for a cocaine-induced condition. The subject is preferably ananimal, including, but not limited to, mammals, reptiles, and avians,more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, andmost preferably human.

An effective amount of the mutant CocE polypeptides described herein isgenerally that which can reduce the cocaine-toxicity or the severity ofa cocaine-induced condition. Reduction in severity includes, forexample, an arrest or a decrease in symptoms, physiological indicators,biochemical markers, or metabolic indicators. When used in the methodsof the invention, a therapeutically effective amount of mutant CocEpolypeptide described herein can be employed in pure form or, where suchforms exist, in pharmaceutically acceptable salt form and with orwithout a pharmaceutically acceptable excipient. For example, the mutantCocE polypeptides of the invention can be administered at a reasonablebenefit/risk ratio applicable to any medical treatment, in an amountsufficient to substantially reduce the cocaine concentration in theblood and/or tissues of the subject.

Toxicity and therapeutic efficacy of mutant CocE polypeptides can bedetermined by standard pharmaceutical procedures in cell cultures and/orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that can be expressed as the ratio LD₅₀/ED₅₀,where large therapeutic indices are preferred.

The amount of mutated CocE polypeptide that can be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses. Agent administration canoccur as a single event or over a time course of treatment. For example,an agent can be administered daily, weekly, bi-weekly, or monthly. Forsome conditions, treatment could extend from several weeks to severalmonths or even a year or more.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including thecocaine-induced condition being treated and the severity of thecocaine-induced condition; activity of the mutant CocE polypeptideemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration;the route of administration; the plasma half-life of the mutant CocEpolypeptide; the rate of excretion of the mutant CocE polypeptideemployed; the duration of the treatment; drugs used in combination orcoincidental with the mutant CocE polypeptide employed; and like factorswell known in the medical arts (see e.g., Koda-Kimble et al. (2004)Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams &Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics,4^(th) ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel(2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton& Lange, ISBN 0071375503). It will be understood by a skilledpractitioner that the total daily usage of the mutant CocE polypeptidefor use in embodiments of the invention disclosed herein will be decidedby the attending physician within the scope of sound medical judgment.

Mutant CocE polypeptides described herein can also be used incombination with other therapeutic modalities. Thus, in addition to thetherapies described herein, one can also provide to the subject othertherapies known to be efficacious for particular cocaine-inducedconditions.

The mutant CocE polypeptides described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers and/or excipients (see e.g., Gennaro (2005) Remington theScience and Practice of Pharmacy 21^(st) ed. Lippincott Williams &Wilkins, ISBN 0781746736). Such formulations will contain atherapeutically effective amount of the mutant CocE polypeptide,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the subject. Theformulation should suit the mode of administration. Mutant CocEpolypeptides of use with the current invention can be formulated byknown methods for administration to a subject using several routes whichinclude, but are not limited to, parenteral, pulmonary, oral, topical,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, ophthalmic, buccal, and rectal. The mutant CocEpolypeptide can also be administered in combination with one or moreadditional agents disclosed herein and/or together with otherbiologically active or biologically inert agents. Such biologicallyactive or inert agents can be in fluid or mechanical communication withthe agent(s) or attached to the agent(s) by ionic, covalent, Van derWaals, hydrophobic, hydrophillic or other physical forces.

Mutant CocE polypeptides described herein can be administeredparenterally, including intravenous, intramuscular, subcutaneous, andintraperitoneal injections. Excipients, commonly used in the parenteraldelivery of small drug molecules, including solubility enhancers,osmotic agents, buffers, and preservatives, can also be included inbiomolecule formulations. Inclusion of antiaggregation andantiadsorption agents, such as surfactants and albumin, when formulatingand delivering biomolecules can add increased stability and decrease therisk of the active biomolecule interacting with an interface, which canlead to unfolding, aggregation, and/or precipitation. The mutant CocEpolypeptide can be lyophilized for added stability during storage, andre-processed before parenteral administration.

Preferably, the mutant CocE polypeptide is pegylated, thus providingincreased stability and lessened immunogenicity (see above).

Pulmonary delivery of macromolecules, such as mutant CocE polypeptides,provide for relatively easy, non-invasive administration to thecirculatory system for systemic distribution (see e.g., Cryan (2004)AAPS J. 7(1) article 4, E20-41, providing a review of pulmonary deliverytechnology). Advantages of pulmonary delivery include noninvasiveness,large surface area for absorption (˜75 m²), thin (˜0.1 to 0.5 μm)alveolar epitheliuem permitting rapid absorption, absence of first passmetabolism, decreased proteolytic activity, rapid onset of action, andhigh bioavailablity. Various inhalation delivery devices, such asmetered-dose inhalers, nebulizers, and dry-powder inhalers, that can beused to deliver the biomolecules described herein are known to the art(e.g., AErx (Aradigm, CA); Respimat (Boehringer, Germany); AeroDose(Aerogen Inc., CA)). Dry powder inhalation devices are particularlypreferred for pulmonary delivery of protein-based agents (e.g.,Spinhaler (Fisons Pharmaceuticals, NY); Rotohaler (GSK, NC); Diskhaler(GSK, NC); Spiros (Dura Pharmaceuticals, CA); Nektar (NektarPharmaceuticals, CA)).

Controlled-release (or sustained-release) preparations can be formulatedto extend the activity of the mutant CocE polypeptide and reduce dosagefrequency. Controlled-release preparations can also be used to effectthe time of onset of action or other characteristics, such as bloodlevels of the agent, and consequently affect the occurrence of sideeffects. Controlled-release preparations can be designed to initiallyrelease an amount of mutant CocE polypeptide that produces the desiredtherapeutic effect, and gradually and continually release other amountsto maintain the level of therapeutic effect over an extended period oftime. In order to maintain a near-constant level of mutant CocEpolypeptide in the body, the agent can be released from the dosage format a rate that will replace the amount of agent being metabolized and/orexcreted from the body. The controlled-release of an agent can bestimulated by various inducers, e.g., change in pH, change intemperature, enzymes, water, or other physiological conditions ormolecules.

Controlled-release systems can include, for example, an infusion pumpwhich can be used to administer the agent in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. The agents of the invention can be administered by othercontrolled-release means or delivery devices that are well known tothose of ordinary skill in the art, including, for example,hydropropylmethyl cellulose, other polymer matrices, gels, permeablemembranes, osmotic systems, depots, multilayer coatings, microparticles,liposomes, microspheres, or the like, or a combination of any of theabove to provide the desired release profile in varying proportions.Other methods of controlled-release delivery of agents will be known tothe skilled artisan and are within the scope of the invention.

Mutant CocE polypeptides can be encapsulated and administered in avariety of carrier delivery systems. Carrier-based systems forbiomolecular agent delivery can: provide for intracellular delivery;tailor biomolecule/agent release rates; increase the proportion ofbiomolecule that reaches its site of action; improve the transport ofthe drug to its site of action; allow colocalized deposition with otheragents or excipients; improve the stability of the agent in vivo;prolong the residence time of the agent at its site of action byreducing clearance; decrease the nonspecific delivery of the agent tonontarget tissues; decrease irritation caused by the agent; decreasetoxicity due to high initial doses of the agent; alter theimmunogenicity of the agent; decrease dosage frequency, improve taste ofthe product; and/or improve shelf life of the product.

Examples of carrier delivery systems for with mutant CocE polypeptidesdescribed herein include microspheres (see e.g., Varde & Pack (2004)Expert Opin. Biol. 4(1) 35-51), hydrogels (see generally, Sakiyama etal. (2001) FASEB J. 15, 1300-1302), polymeric implants (see generally,Teng et al (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 3024-3029), smartpolymeric carriers (see generally, Stayton et al. (2005) OrthodCraniofacial Res 8, 219-225; Wu et al. (2005) Nature Biotech (2005)23(9), 1137-1146), and liposomes (see e.g., Galovic et al. (2002) Eur.J. Pharm. Sci. 15, 441-448; Wagner et al. (2002) J. Liposome Res. 12,259-270). Preferably, the mutant CocE polypeptide is encapsulated in RBC(see above; Example 12).

Screening Methods

Another aspect of the invention is directed toward screening methods forthe generation, identification, and purification of thermostable mutantCocE polypeptides. Generally, mutant CocE polypeptides can be initiallydesigned according to the approaches described above. Such designedpolypeptides can then be screened for preferred characteristics, such asretention of hydrolytic efficiency, increased thermostability, increasedplasma-half-life, and/or reduced antigenicity. Also, random mutant CocEpolypeptides can be screened for the desired characteristics.

Detection methods to screen for thermostable mutants encompass a widevariety of techniques. The following is an exemplary summary of ageneric protocol. Nucleic acid encoding the mutant CocE polypeptides(generated, for example, through rational design, random mutagenesis, orhost mutagensis) is transformed into an appropriate expression host (forexample, E. coli cells such as E. coli BL21 Gold (Stratagene)), andexpression of the mutant polypeptide is induced according to standardprotocols (e.g. by IPTG). Expression is performed at temperatures toproduce optimal protein expression (e.g., 16° C. for CocE, see e.g.,Example 1) for a pre-determined period of time (e.g., anywhere from 30minutes to 24 hours or longer). Alternatively, expression is performedat an elevated temperature (for example, the elevated temperature can beat least about 35° C., at least about 36° C., at least about 37° C., atleast about 38° C., at least about 39° C., at least about 40° C., oreven higher). Preferably, the elevated temperature at which mutated CocEpolypeptide is expressed is 37° C. Around this temperature, wild-typeCocE polypeptide partitions almost exclusively into inclusion bodies.Cells containing the expressed mutant polypeptides are screened for thepresence of thermostable variants of CocE.

Screening for the presence of thermostable variants of CocE generallyinvolves either direct measurement on cultured cells, on cell lysates,or following cellular disruption and isolation of the mutant CocEpolypeptide. Cellular disruption can include osmotic shock, chemicallysis, soniciation, and/or homogenization, and isolation of the mutantpolypeptide can be obtained through numerous methods including eitherdirect absorption to a matrix or affinity absorption through the use ofanti-cocaine antibodies or fusion-protein specific capture systems.Suitable matrix for absorption includes nitrocellulose paper, filters,untreated or affinity-treated microtiter plates, agarose or sepharoseresins, and/or affinity-coated tips.

The esterase activity of the cultured cells or isolated mutantpolypeptide can be subsequently measured at one or more temperatures todetermine the thermostability of the mutants. The temperature at whichthe activity assay is performed determines the degree of thermostabilitydetection. Thus, while the final mutants will preferably have a meltingtemperature of 45° C. or higher (as determined by, for example, circulardichroism), often, the initial screening at 45° C. will not find activeenzymes. Rather, several cycles of mutagenesis and screening atsubsequently increasing temperatures can be performed to achievethermostable mutants. Thus, initial screening can be performed at 30°C., and after further cycles of mutagenesis, screening can be performedwith incrementally increasing temperatures (for example, 34° C., 37° C.,40° C., 42.5° C., 45° C., etc.), until a mutant of suitablethermostability is achieved. The incremental temperature increases aredetermined empirically during the procedure, and are affected by thenumber of hits at particular temperatures and the determined Tm of thegenerated mutants.

While under no obligation to do so, and while not wishing to be bound bytheory, herein follows what is believed to be a mechanistic explanationof melting of various embodiments of mutant CocE polypeptides describedherein. CD spectra data illustrate that the CD melting of cocaineesterase and mutants is irreversible, as cooling to 0 degrees does notreform the original spectra (see, e.g., Example 19). While thermodynamicparameters cannot be ascertained, CD spectra can be used tocomparatively determine whether mutants are more or less stable, whetherthey have different secondary structures or aggregation properties. Itis believed that the CocE polypeptides described herein melt via anintermediate step, that is, the polypeptides undergo a 2-step meltingprocess.

Detection of esterase activity can be performed using a variety ofmethods, where substrates are coupled to a specific detection system.Appropriate substrates for use in determining esterase activity caninclude cocaine, tritiated (3H) cocaine, cocaine substrate derivativessuch as a thio-cocaine derivative (see e.g., FIG. 6), and/or substratesthat report general esterase activity such as 4-nitrophenyl acetate. Thedetection system can be directly coupled to the specifics of thesubstrate, for example: cleavage of unmodified cocaine can be detectedby monitoring changes in cocaine absorbance at 240 nm (see e.g., Example4), or by monitoring pH changes that result from the accumulation of theacidic benzoic acid product (see e.g., Example 15), or through the useof cocaine aptamers (see e.g., Stojanovic, M. N., de Prada, P. & Landry,D. W. (2001) J Am Chem Soc 123, 4928-4931; Stojanovic, M. N. & Landry,D. W. (2002) J Am Chem Soc 124, 9678-9679) by monitoring changes influorescence upon degradation of cocaine (see example 15); cleavage oftritiated (3H) cocaine can be detected by acidification and detection oftritiated benzoic acid product through separation by chromatography (seeexample 1 and 15); cleavage of cocaine derivatives such as thio-cocainecan be monitored by the detection of reactive sulfhydryl groups, throughthe addition of Ellman's reagent and determination of absorbance changesat 412 nm (see e.g., Example 15), or by the addition and visualizationof precipitating sulfhydryl reacting heavy metals; cleavage of4-nitrophenyl acetate can be detected by monitoring changes inabsorbance at 420 nm (see e.g., Halgasova, N. et al. (1994) Biochem J298 Pt 3, 751-755; O'Conner, C. J. & Manuel, R. D. (1993) J Dairy Sci.76, 3674-3682).

Mutant CocE polypeptides identified through the above procedures, or asimilar high throughput assay, can be further evaluated using in vitroprocedures described herein (e.g., Kcat and Km values, stability at 37°,melting temperature (Tm), endotoxin levels, ability to degrade cocainein plasma). Mutant CocE polypeptides with thermostable esterase activityand/or reduced immunogenicity, can be further evaluated using in vivoprocedures described herein (e.g., potency, duration of action, effectswith repeated dosing, and/or immunological evaluation). Preferably,mutant CocE polypeptides magnitudinal decrease of cocaine toxicity isexamined first (see e.g., Examples 5, 7, and 8), and those mutants thatreduce toxicity by at least about 5-10 fold can be further evaluated fortime course of action (see e.g., Example 6). Candidate mutant CocEpolypeptides can be further stabilized by, for example, pegylationand/or encapsulation in RBC and re-evaluated in the above describedprocedures.

Having described the invention in detail, it will be apparent thatmodifications, variations, and equivalent embodiments are possiblewithout departing the scope of the invention defined in the appendedclaims. Furthermore, it should be appreciated that all examples in thepresent disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratevarious embodiments of the invention disclosed herein. It should beappreciated by those of skill in the art that the techniques disclosedin the examples that follow represent approaches the inventors havefound function well in the practice of the invention, and thus can beconsidered to constitute examples of modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 CocE Expression

A method for expression and purification of CocE was established, whereCocE is expressed in E. Coli as a fusion protein with a carboxylterminus His×6-tag. The cocaine esterase gene was subcloned into the E.coli expression vector pET-22b(+) and high levels of cocaine esteraseenzyme containing a C-terminal histidine tag were induced upon theaddition of IPTG at 23 C. The recombinant protein accumulated toapproximately 10-15% of total protein. CocE was enriched on either acobalt-chelate column (Talon™ Clontech) or a nickel-chelated agarosecolumn (Pierce) by virtue of the His×6 tag. The eluted protein wasapproximately 95% pure by SDS-PAGE and Coomassie blue staining, and wassubsequently resolved by ion-exchange chromatography FPLC column(Q-Sepharose) using an NaCl gradient. The protein eluted as a singlepeak was approximately 99% pure, as indexed by SDS-PAGE and Coomassieblue staining (see e.g., FIG. 3).

Enzyme activity was determined through two assays: a radio-ligandactivity assay in which tritated cocaine was hydrolyzed and then afteracidification, the tritiated benzoic acid product was separated fromtritiated cocaine hydrochloride by chromatography; and aspectrophotometric assay under similar conditions as described by Turneret al. (2002) Biochemistry 41, 12297-12307. The unique absorptionspectra of cocaine (extinction coefficient 6.7 L/mmol/cm, at 240 nm)allows for the observation of remaining cocaine following enzymaticcleavage. The initial linear rates of decay of cocaine, representingvelocity, were determined in a SpectraMax 190 plate reader (MolecularDevices) using SOFTmax Pro software (v1.13). The reaction was initiatedby adding 150 μL of a 2× enzyme solution to the 150 μL of a 2× cocainesolution. Final CocE concentrations ranged from 100 ng/mL to 20 ng/mL.Final cocaine concentrations were as follows: 250, 126, 62.5, 31.25,15.63, 7.81, 3.91, and 1.95 μM. For the kinetics of all enzymes, thebuffer was phosphate buffered saline, pH 7.4. Initial rates were fittedto the Michaelis-Menten equation, with kcat and Km as adjustableparameters (GraphPad; PRISM, v4).

As determined using the cocaine spectrophotometric assay, the purifiedwild-type CocE polypeptide hydrolyzes cocaine with a Kcat of approx. 500min⁻¹ and a Km of approx. 2 μM, which is consistent with previouslyreported values (see e.g., Turner et al. 2000).

Such expression procedures can be utilized for the mutant CocEpolypeptides described herein.

Example 2 Ex Vivo CocE Plasma Activity

Ex vivo determination of cocaine levels following cocaine esterase wasexamined in human plasma (University of Michigan Hospital blood bank).Cocaine was obtained from The National Institute of Drug Abuse(Bethesda, Md., USA). Cocaine was dissolved in sterile water. Aliquots(3 ml) of human plasma were maintained at 37° C. in a water bath for 10minutes prior to the start and for the duration of the experiment. Afterequilibrating plasma in water bath, cocaine was added to a finalconcentration of 300 μM and vortexed for 30 seconds. Plasma samples wereremoved and placed in a microcentrifuge tube containing the internalstandard and a saturated sodium fluoride solution to prevent furthercocaine metabolism. Immediately after taking the first plasma ample(cocaine alone), 0.05 mg/ml CocE or vehicle CocE was added and vortexed.Plasma samples were collected at 1, 2, 4, 6, 8, 10, 15, 30, 45, 60, and120 minutes after adding CocE. Levels of cocaine were measured usinghigh performance liquid chromatography tandem mass spectrometry.

Liquid chromotography was performed using a Surveyor HPLC system(ThermoElectron Corp., Franklin, Mass.) with a quanternary pump andautosampler configured with a 10 μl injection loop. Separation wasachieved using a Phenomex C18 3 μm 30×4.6 mm column with correspondingguard column (Waters Corp., Milford, Mass.) at a flow rate of 600μl/min. Solvent A consisted of a 0.1% formic acid solution, and solventB was 0.1% formic acid in acetonitrile (high purity grade; Burdick andJackson, Muskegon, Mich.). A 3 minute ballistic gradient was used withcocaine and the internal standard co-eluting at 2.3 min.

For detection and quantification by mass spectrometry, a Finnigan TSQQuantum Ultra AM triple quadrapole mass spectrometer equipped with anIonMax electrospray ionization source (ThermoElectron Corp., Franklin,Mass.) was used in positive ion, selected reaction monitoring mode.Nitrogen served as the nebulizing gas and argon as the collision gas.Gas flow rates, spray voltages, and collision energies were optimized.Calibration curves were determined for cocaine with 50 nM deuteratedcocaine (cocaine D₃) as the internal standard in untreated plasmasamples. Unknown samples were also spiked with cocaine D₃. All sampleswere evaluated in triplicate. Standard curves and unknowns were analyzedby Quan Browser program in Xcalibur version 1.4 (ThermoElectron Corp.,Franklin, Mass.) software. Calibration curves were constructed usinglinear regression of cocaine peak area/internal standard area ratio as afunction of standard concentration with a weighting factor of 1/x.Standard curve fit values were accepted at a value is greater than 0.99,and RSD values for replicate samples are between 0-10%.

Results demonstrate that CocE is able to degrade cocaine very rapidlywhen the two are mixed briefly in human plasma (see e.g., FIG. 4). Thefirst time point indicates the cocaine concentration prior to theaddition of cocaine esterase or esterase vehicle. Prior to esterasetreatment, cocaine levels were similar, but within 1 min of cocaineesterase administration, cocaine levels were decreased at least 100-foldto approximately 2 μM as compared with the vehicle-treated plasmasample. Cocaine levels continued to decrease in the esterase-treatedplasma samples, falling below 1 microM by the 2-minute timepoint.

The therapeutic efficiency of the enzyme was demonstrated by theincreasing dose of cocaine required to produce toxic effects after asingle intravenous injection of CocE. The wild-type enzyme demonstratedrapid kinetics for cocaine degradation ex vivo in rat and human serum.Two inactive mutants of CocE failed to protect the rats from the toxiceffects of cocaine, confirming the protective effects are due tohydrolytic activity. Furthermore, CocE did not change the lethality ofWIN-35065-2, a cocaine analog that lacks the benzoyl ester moietytargeted by CocE. The in vivo and ex vivo characterization of CocEsupports the role of the enzyme as a suitable antidote to toxicity inhumans.

Example 3 Predicted Thermostable Mutant CocE

Rational design of mutant CocE described herein was based upon moleculardynamics (MD) simulations. A computational model of CocE was constructedusing the published crystal structure of wild-type CocE (see e.g., FIG.2). Such models can be used to identify certain amino acid modificationsthat increase the theoretical melting temperature of the protein withoutdisrupting the structure at the active site. Classical MD simulationenables the study of time evolution of a large system by taking manysmall successive time steps under atomic forces determined by a set ofparameterized interaction functions (force field), including bondedinteractions (bonds, angles, and dihedral angles), non-bonded van derWaals interactions, and electrostatic interactions based or net atomiccharges. Due to the simple force field form the MD simulation may beperformed for a sufficiently long simulation time to give meaningfulensemble-averaged properties, even for a very large system involvingover a hundred thousand atoms. So, for CocE and each proposed mutant,the MD simulation will lead to a reasonable, dynamically averaged 3Dstructure of the simulated polypeptide in water.

Based on the X-ray crystal structure (PDB code 1JU3) of the bacterialcocaine esterase (CocE) (Larson et al. (2002) Nature 9, 17), a complete3D model of CocE binding with (−)-cocaine suitable for computationalmodeling its thermodynamic stability was built. To increase thethermostability of CocE, a computational method was implemented inRosettaDesign program (Kuhlman and Baker (2000) PNAS 97, 10383) capableof predicting thermostabilizing mutations within a given fold whileminimizing any shift in the backbone that might structurally disrupt theactive site structure or quench its flexibility. The method implementedin the RosettaDesign program uses an energy function for evaluating thefitness of a particular sequence for a given fold and a Monte Carlosearch algorithm for sampling sequence space. A similar method has beensuccessfully used by other researchers to increase thermostability of anenzyme with no reduction in catalytic efficiency (Korkegian et al.(2005) Science 308, 857). The partial atomic charges for thenon-standard residue atoms were calculated by using the standard RESPprotocol implemented in the Antechamber module of the Amber7 (or 8)program package (Case, 2002). The computational modeling using theRosettaDesign program allowed prediction of a set of CocE mutationscalculated as having lower energy and, therefore, increasedthermostability (see e.g., Table 2). For this example, computationconsidered only possible mutations on the amino acid residues having adistance of between 6-25 {acute over (Å)} from the cocaine substratemolecule.

Identified single mutation CocE polypeptides calculated to stabilizeCocE by about 2.1 to about 4.5 kcal/mol included: L163V (SEQ ID NO: 3);V121D (SEQ ID NO: 4); S167A (SEQ ID NO: 5); Q123E (SEQ ID NO: 6); V225I(SEQ ID NO: 7); I218L (SEQ ID NO: 8); A310D (SEQ ID NO: 9); A149S (SEQID NO: 10); S159A (SEQ ID NO: 11); S265A (SEQ ID NO: 12); S56G (SEQ IDNO: 13); W220A (SEQ ID NO: 14); T122A (SEQ ID NO: 15); S140A (SEQ ID NO:16); F189L (SEQ ID NO: 17); A193D (SEQ ID NO: 18); T254R (SEQ ID NO:19); N42V (SEQ ID NO: 20); V262L (SEQ ID NO: 21); L508G (SEQ ID NO: 22);Y152H (SEQ ID NO: 23); V160A (SEQ ID NO: 24); T172R (SEQ ID NO: 25);Y532F (SEQ ID NO: 26); T74S (SEQ ID NO: 27); W285T (SEQ ID NO: 28);L146P (SEQ ID NO: 29); D533S (SEQ ID NO: 30); A194R (SEQ ID NO: 31);G173Q (SEQ ID NO: 32); C477T (SEQ ID NO: 33); K531A (SEQ ID NO: 34);R41I (SEQ ID NO: 35); L119A (SEQ ID NO: 36); K46A (SEQ ID NO: 37); andF84Y(SEQ ID NO: 38)

TABLE 2 Summary of computational modeling using the RosettaDesignprogram with consensus approach Energy Consensus Change R1 R2 R3 R4 R5R6 R7 R8 R9 R10 mutation (kcal/mol) 163 163 163 163 163 163 163 163 163163 L163V −4.5 121 121 121 121 121 121 121 121 121 121 V121D −3.9 167167 167 167 167 167 167 167 167 167 S167A −3.9 123 123 123 123 123 123123 123 123 123 Q123E −3.8 225 310 310 225 225 225 225 310 225 218 V225I−3.6 218 225 218 218 218 218 218 225 218 225 I218L −3.5 310 218 225 310310 310 310 218 310 152 A310D −3.4 149 149 152 149 149 149 149 149 149310 A149S −3.3 159 159 140 159 159 159 159 159 159 149 S159A −3.3 189265 149 265 265 265 265 265 189 265 S265A −3.3 265 140 265 56 56 56 56220 265 159 S56G −3.2 56 220 159 220 220 220 220 122 56 220 W220A −3.2220 122 220 122 122 122 122 140 220 56 T122A −3.1 122 189 122 140 140140 140 189 122 122 S140A −3.1 140 193 189 189 189 189 189 193 140 140F189L −3.1 254 42 193 193 193 193 193 42 254 189 A193D −3.1 262 262 42254 254 254 254 254 42 193 T254R −3.1 508 508 262 42 42 42 42 262 262262 N42V −3.0 152 152 508 262 262 262 262 508 508 508 V262L −3.0 160 160198 508 508 508 508 152 152 198 L508G −2.9 74 198 160 152 152 152 152160 160 254 Y152H −2.9 172 74 74 160 160 160 160 198 198 74 V160A −2.8193 172 172 198 198 74 198 74 172 160 T172R −2.8 532 532 532 532 74 17274 172 74 172 Y532F −2.7 146 146 56 74 172 532 172 532 193 532 T74S −2.7285 285 285 172 532 146 532 146 532 285 W285T −2.6 533 290 146 285 146285 146 285 285 290 L146P −2.6 173 254 533 146 285 533 285 533 146 146194 533 173 533 533 173 533 173 533 533 D533S −2.5 477 56 194 173 194194 173 194 173 173 A194R −2.4 531 173 200 194 200 290 194 200 194 194G173Q −2.4 42 194 477 200 290 477 200 290 200 200 C477T −2.4 119 200 531290 477 531 477 477 290 477 K531A −2.4 200 477 305 477 531 200 531 531477 531 41 531 41 41 41 41 41 41 531 42 R41I −2.2 46 41 119 119 119 119119 119 41 119 L119A −2.2 84 119 46 46 173 46 46 46 119 41 K46A −2.1 30546 84 84 46 84 84 56 46 46 F84Y −2.1 478 57 158 478 84 478 305 84 84 5757 84 307 57 478 57 478 307 478 84 87 478 478 142 57 87 57 478 57 158142 87 57 263 87 142 87 57 142 478 263 142 142 307 142 263 142 142 263142 307 263 263 78 263 307 263 263 307 263 78 307 78 257 307 78 307 7878 78 257 78 257 531 48 257 78 257 257 257 290 257 290 49 78 49 257 4949 49 291 201 49 201 257 305 201 201 201 201 49 49 201 305 45 291 290412 305 307 176 291 291 412 49 176 291 291 412 291 45 305 412 291 201 4549 176 291 176 54 176 176 176 305 54 176 45 45 305 406 45 254 45 291 40645 413 176 45 50 54 45 413 176 50 54 54 413 54 406 54 54 54 406 305 54406 50 413 50 406 50 406 406 50 406 406 50 50 50 50

Example 4 Kinetic Parameters of T172R and S159A Mutant CocE

Wild-type CocE and the T172R and S159A mutant CocE polypeptides weretested for catalytic efficiency.

Site directed mutagenesis (QuickChange™, Invitrogen) of CocE wasperformed to generate the S159A (SEQ ID NO: 11) mutant CocE polypeptide.Cloning and expression techniques used to produce the S159A were thesame as in Example 1, except as indicated otherwise. The CocE gene wasamplified through Polymerase Chain Reaction (PCR) in the presence ofprimers that contain the specific mutation required (Integrated DNATechnologies, Inc.). The specific mutation was subcloned back into theexpression plasmid and the nucleotide sequence of these plasmidsdetermined to verify the presence of the mutation.

Mutant T172R was generated by overlapping PCR using 5′ and 3′ primerscontaining the specific T172R mutation, as well as an additional Sac IIrestriction enzyme site for easy detection of the mutated gene. Primerpairs CocE 20-5′F-Nde I (5′ GATATACATATGGTGGACGGGAATTAC 3′) andT172R-3′R (5′ CAGACCTCGACGTGATGAGCCCGCGGCCTATGAGAGCTGACCAGC 3′) as wellas CocE-1800-3′R (5′ GTGGTGCTCGAGTCGCTTGATAATCG 3′) and T172R-5′F (5′GCTGGTCAGCTCTCATAGGCCGCGGGCTCATCACGTCGAGGTCTG 3′) were PCR-amplifiedusing the high-fidelity Pfu enzyme (Stratagene) with an annealingtemperature of 55° C. Resultant PCR products were combined andre-amplified, generating a full-length CocE gene encoding the T172Rmutation. The gene was digested with Nde I and Xho I; subcloned into theexpression vector, and sequenced in its entirety to verify both thepresence of the mutation and the absence of additional PCR-copy errormutations.

Plasmids containing the mutations were transformed into E. coli BL21cells and IPTG-induced enzyme was purified on Ni-Agarose. Expressedproteins were then tested for enzymatic activity and for thermostabilityat 37° C. Enzyme activity was measured using the spectrophotometricassay as described in Example 1. Thermostability was also tested via thespectrophotometric assay by pre-incubation of both wild-type and mutantsat 37° C. for various times. Additionally, the nature of thethermo-instability of both the wild-type and T172R mutant was analyzedby polyacrylamide gel electrophoresis under denaturing andnon-denaturing conditions. Briefly, mutant and wild-type enzyme at 0.1mg/ml were incubated at 37° C. for various time-points, cooled to 4° C.,mixed with SDS-loading dye containing-mercaptoethanol and run on 10%SDS-PAGE gels (denaturing conditions) or run on native 10%polyacrylamide gels (non-denaturing conditions) at 4° C. Gels were fixedwith 10% methanol, 7% acetic acid for 30 minutes, and then stained withSypro-Ruby protein gel stain (Molecular Probes, Invitrogen) for 3 hours.Protein staining was visualized under UV-light using an AlphaImager™3400 (Alpha Innotech). Finally, the exact melting temperature of boththe wild-type and T172R mutant were determined by circular dichroismusing a JASCO-810 spectropolarimeter driven by a JASCO V500/FP-750analysis program for Windows. The CD spectra were measured inmillidegrees and normalized against PBS buffer.

Results from the spectrophotometric assay showed that T172R had anincreased Vmax and Km at room temperature and a greatly increased Vmaxand Km at 37° C. as compared to wild-type CocE (see e.g., Table 3; FIG.5). In fact, the T172R Vmax and Km at 37° C. was comparable to the Vmaxand Km of wild-type CocE at room temperature. The S159A mutant CocEshowed a slightly increased Vmax and Km at 37° C., but a decreased Vmaxand Km at room temperature, as compared to wild-type CocE.Electrophoresis under denaturing conditions (FIG. 6 a) indicated asingle protein band for both the wild-type and T172R of approximately65000 Da, regardless of incubation temperature, indicating theproteolytic degradation does not adequately describe the mechanism forthermoinstability. The non-denaturing gels (FIG. 6 b) showed a singleprotein band for the wild-type enzyme prior to incubation at 37° C.,however upon incubation at 37° C., higher molecular weight species areseen to appear as the original band fades. These putative proteinaggregates can also be observed for the T172R mutant, however the timeto aggregation upon 37° C. incubation is longer, and in this procedurethe T172R mutant was approximated to have a 8× longer half-life at 37°C. than the wildtype. Analysis of the protein melting temperature bycircular dichrosim (FIG. 7) indicated temperature sensitive changes inprotein tertiary structure were occurring in the near-UV range of thespectrum (between 260 nm and 320 nm). Curve-fitting oftemperature-sensitive changes in this region of the spectrum indicatedthe wild-type CocE has a melting temperature of 36.15° C., withdetectable denaturation beginning at approximately 30° C. The T172Rmutant was determined to have a melting temperature of 41.43° C., withdetectable denaturation beginning at approximately 28° C. Thus thesingle amino acid change from Tyrosine to Arginine at Amino acid 172,with an estimated 2.8 kCa/mol increase in thermostability, wasdetermined to have a full 5 degrees increased melting temperaturecompared to the wild-type CocE.

TABLE 3 Kinetic parameters for T172R, S159A, and wt CocE. S159A T172R wtCocE (40 min (40 min@ wt (40 min@ S159A @ 37° C.) T172R 37° C.) CocE 37°C.) Vmax 876.6 130.7 1466 1267 1264 94.06 Km 43.65 15.69 88.20 78.8071.81 12.30

Example 5 In Vivo Wild-Type CocE Prevention of Cocaine Lethality in Rats

To determine CocE's esteratic activity in vivo, a rodent model of acutecocaine toxicity was implemented. When treated with high doses ofcocaine, rats first exhibit convulsions followed by cessation ofrespiration and movement. The lowest toxic dose of cocaine, whenadministered intraperitoneally, will produce fatality within 15 minutesof treatment.

Protection against cocaine-induced lethality by wild-type CocE wasdetermined and compared to the protective effects of human BChE. Theesteratic activity of CocE was established by assessing the activity oftwo mutant enzymes, each lacking one of three amino acids in the activesite. Additionally, activity of a modified wild-type enzyme by acovalent modification of Ser17 within the active site, by phenylmethylsulphonate fluoride (PMSF) was determined. Esteratic degradation ofcocaine was shown to be the mechanism of CocE's protective effects byverifying if the enzyme protected against toxicity induced by WIN35065-2 (Madras et al. (1989) J Pharmacol Exp Ther 251, 13-141), acocaine analog which lacks the ester bridge at the proposed site ofenzymatic hydrolysis.

Male Sprague-Dawley rats (300 grams) (Harlan Sprague Dawley,Indianapolis, Ind.) were housed three animals per cage. Followingsurgical implantation of a jugular catheter, all rats were individuallyhoused until termination of the experiment. Rats were maintained on a12-h light/dark cycle, with lights turned on at 7:30 a.m. and food andwater were available ad libitum. After rats were anesthetized withketamine hydrochloride (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.),intravenous catheters (Micro-renethane tubing, 15 cm, MRE-040, BraintreeScientific Inc., Braintree, Mass.) were implanted into the right jugularvein. Approximately 3 cm of the catheter was inserted into the vein; theremaining tubing was passed subcutaneously to the back, where it exitedfrom an incision made between the shoulder blades. The exposed tubingwas capped with a 1 cm piece of stainless steel (0.28 diameter, SmallParts Inc., Miami, Fla.). Catheters were flushed daily with 0.5 ml ofheparinized saline (50 U/ml) to maintain catheter patency. Followingsurgery, rats were allowed one week to recover. Each rat was used for asingle experiment, and all experimental groups consisted of 6-8 rats.

To determine the lowest effective dose of CocE that blockedcocaine-induced convulsions and death, 0.1, 0.32, or 1.0 mg CocE orvehicle (phosphate buffered saline, PBS) was administered intravenouslyone minute after 180 mg/kg cocaine (i.p.). To determine the catalyticlimits of CocE, increasing doses of cocaine were administered (100, 560,1000 mg/kg, i.p.) one minute prior to 1.0 mg CocE (i.v.). Mutants andPMSF-blocked CocE were administered (1 mg, i.v.) one minute before 180mg/kg cocaine (i.p.). CocE (1.0 mg, i.v.) was also administered oneminute after the lowest dose of WIN-35065-2 (560 mg/kg, i.p.). CocE (1.0mg, i.v.) was given before and after cocaine (100 mg/kg, i.p.) todetermine the in vivo half-life of the esterase. All intravenousinjections were followed by a heparinized saline flush (0.5 ml). Aftertreatment, rats were observed for convulsion; and death. Number ofconvulsant episodes, duration of each episode, and type of convulsionwere recorded. Death was defined as cessation of observed movement andrespiration. Percent of animals in each experimental group exhibitingconvulsions and lethality were calculated. Percent standard error meanwas then calculated for each data point.

Results showed that, in the rodent model of acute toxicity, cocainedose-dependently induced convulsions and death in rats; death wasobserved in less than 15 minutes after administration in 100% of animalsgiven 100 mg/kg cocaine (see e.g., FIG. 9). CocE (1.0 mg) infused aftercocaine administration produced a ten-fold shift in the cocaine-toxicitydose effect curve (see e.g., FIG. 9), such that 1000 mg/kg cocaine wasrequired to surmount the protective, catalytic properties of CocE. Thistreatment regimen closely resembles human toxicity situations, where theantidote to overdose is given only after cocaine has been ingested,inhaled, or injected.

Wild-type CocE showed superior catalytic efficiency over human BChE.Given one minute before 180 mg/kg cocaine, 1 mg CocE offered 100%protection against cocaine-induced lethality (see e.g., FIG. 10), whilea 10-times molar equivalent dose of human BChE (13 mg) offered noprotection, similar to a ten-fold lower dose of CocE (0.1 mg) (see e.g.,FIG. 10).

Both mutations of CocE (Ser117Ala or Tyr44Phe) lacked in vivo activityand, therefore, had no protective effects (FIG. 11 b). Furthermore,PMSF-treated enzyme also eliminated CocE's protective effect againstcocaine (FIG. 11 b). Additionally, the lethal effect of thenonhydrolyzable cocaine analog, WIN 35065-2, was not overcome bytreatment with CocE (FIG. 12). Based upon in vivo protection studiesperformed with catalytically inactivated preparations of the enzyme(PMSF-treated and CocE inactive mutants), it is clear that theprotective effects of the enzyme are due to its ability to hydrolyzecocaine. Taken together, these data comply with in vitro assessments ofCocE's esteratic activity and confirm the enzyme's mechanism ofprotection against cocaine-induced lethality in vivo.

Example 6 Time-Dependent Effects of Wild-Type CocE

The effects of administering wild-type CocE prior to cocaine dosage wasexamine. The rat toxicity model was as described in Example 5. Wild-typeCocE was adminstered 100, 30, 10, 3, and 1 minute before and 1 and 6minutes after cocaine administration. Compound extractions from humanplasma samples were performed in 100% acetronitrile (3× volume),incubated for approximately 15 min, centrifuged at 13,000 rpm for 4.5min, and the resulting supernantant was collected. The extracts wereconcentrated on a Savant Speed Vac (ThermoElectron Corp., Franklin,Mass.) to remove the acetonitrile. Extracted samples were reconstitutedin water and further diluted 10-1000 times.

Human plasma samples were spiked with 300 μM cocaine and maintained at37° C. One aliquot of plasma was sampled prior to the addition ofcocaine esterase or esterase vehicle, and another aliquots was collected1 min following esterase administration. Plasma aliquots were mixedimmediately with the internal standard (cocaine-D₃) and a saturatedsodium fluoride solution to prevent further cocaine metabolism. Tissueextractions were performed and levels of cocaine and internal standardwere quantified by HPLC with tandem mass spectrometry.

For time-dependent inactivation of CocE in vitro, purified CocE enzyme(at 250 ng/ml) was incubated in assay buffer in the absence of cocaineat 37° C. for various times. Following incubation at 37° C. the sampleswere placed on ice. To assess the effect of temperature on CocEactivity, samples were incubated with (−) cocaine at variousconcentrations as indicated at a final enzyme concentration of 125ng/ml. The rate of decay of (−)-cocaine at A240 was measured on amultiplate reader. The data were fitted to a single exponential decayusing Kaleidagraph™ (Synergy software) yielding a t_(i)n of 13.2.

Wild-type CocE was found to have time-dependent protective effects; 100%of rats were saved when treated with CocE (1 mg) 1 minute beforecocaine, while only 66% and 32% of rats survived when treated with CocE30 and 1 minutes before cocaine, respectively (see e.g., FIG. 14).CocE's protective effects were eliminated when rats were treated 100 minprior to cocaine. This time-dependent effect is most likely due to thethermal deactivation of the enzyme in vivo. In rat plasma, CocE wasfound to have a remarkably short half-life (about 10 minutes, FIG. 13),most likely due to sensitivity to changes in pH and temperature.Additionally, preliminary in vitro data with the purified enzymesuggests that CocE undergoes a temperature-dependent inactivation with at_(1/2) of approximately 15 min at 37° C.

Given these data, it can be approximated that a 1 mg of CocE doseadministered 30 minutes prior to cocaine, will decay approximately 3half-lives, leaving 0.25 mg of wild-type enzyme in the generalcirculation when cocaine is administered.

Previous studies have suggested that lethal blood concentrations ofcocaine in the rat vary between 50-128 μM (Mets B and Virag L (1995)Anesth Analg 81, 1033-1038; Mets et al. (1999) Life Sci 65, 1317-1328),and peak plasma levels of cocaine occur about 13 minutes after anintraperitoneal injection (Sun et al. (2002) J Pharmacol Exp Ther 302,710-716). Based on reported kinetics of intraperitoneal cocaineadministration (Sun et al. (2002) J Pharmacol Exp Ther 302, 710-716), itis estimated that 100 and 320 mg/kg cocaine yield peak cocaine bloodconcentration of 35 μM and 113 μM, respectively. Lethal concentrationsof cocaine are of a similar magnitude in humans (20-200 μM) (Finkle B Sand McCloskey K L (1978) J Forensic Sci 23, 173-189; Wetli and Wright(1979) J Am Med Assoc 241, 2519-2522). As 1 mg of wild-type CocE savedrats treated with these doses of cocaine (FIG. 9), it can be justifiablypredicted that the enzyme would protect against cocaine-toxicity in thehuman. Furthermore, cocaine levels measured using high performanceliquid chromatography tandem mass spectrometry in human plasma spikedwith 300 μM cocaine, a concentration that exceeds reported toxic levelsof cocaine, and then treated with CocE (a molar equivalent of our invivo 1.0 mg dose), reduced the cocaine concentration to approximately 2μM in less than a minute (FIG. 10).

Protection against an LD50 dose of cocaine in rats requires a 10 mg/kgtreatment of BChE (Lynch et al. (1997) Toxicol Appl Pharmacol 145,363-371), assuming that the enzyme is distributed similarly in thehuman, a 70 kg individual would require a 700 mg dose of exogenous BChEto protect against an overdose. Furthermore, there is no evidence thatBChE can act to reverse cocaine-toxicity when administered aftercocaine, a necessary characteristic of an antidote for cocaine toxicity.

It is thus demonstrated that a dose of 1 mg wild-type CocE in a 300 gramrat is sufficient to protect against a dose of cocaine that exceeds theLD100 (FIG. 9). Additionally, the enzyme given both before, and moreimportantly, up to 6 minutes after the LD100 dose of cocaine, providedprotection from toxicity (FIG. 14). CocE metabolized cocaineconcentrations in serum by 150-fold in less than one minute (FIG. 15).

Given these data, it is predicted that a 250 mg of CocE administered toa 70 kg human after toxic cocaine ingestion would rescue the individualfrom certain death. The above demonstrates that wild-type CocE is anefficient anti-cocaine molecule but that the short activity time of theenzyme under physiological conditions limits it therapeutic value. Suchresults point to the importance of extending the thermostability of thewild-type CocE.

Example 7 In Vivo T172R Mutant CocE Prevention of Cocaine Lethality inRats

The hydrolytic activity of wild-type CocE and T172R mutant CocE wascharacterized and confirmed in vivo by assessing its ability to preventcocaine-induced lethality in rats.

Animal treatment was as described in Example 5. Increasing doses ofcocaine were administered (i.p.) to rats, and one minute after,wild-type CocE (0.32 mg), mutant CocE T172R (0.32 mg), or vehicle wasadministered intravenously. All intravenous injections were followed bya heparinized flush (0.5 ml). After treatment, rats were observed fordeath. Time until death was recorded for the 1 g/kg cocaine dosage.Wild-type and T172R CocE (0.32 mg) was administered at various timespreceding (1, 10, 30, and 60 minutes) administration of 320 mg/kp i.p.of cocaine, with rats then monitored for death.

Results demonstrate the ability of intravenous CocE and, especially,mutant CocE to reverse or prevent the lethal effects produced bycocaine. Wild-type CocE adminstered one minute after cocaineadministration prevented lethality at cocaine dosages that killedcontrol rats and 100% lethality did not occur until cocaine dosages of 1g/kg (see e.g., FIG. 16). The T172R mutant CocE treated rats were ableto tolerate even higher dosages, with the 1 g/kg of cocaine resulting inonly about 70% lethality. And the prophylactic effects of T172R mutantCocE was longer lasting than the wild-type CocE (see e.g., FIG. 17).

Example 8 In Vivo Repeated CocE Dose in Mice

Additional studies were done on the effect of CocE on toxicity in themouse, particularly with respect to repeated doses.

The animal toxicity model was similar to that described previously,except as described. Male NIH Swiss mice were used. For tail intravenousinjection, mice are placed in a small restraint chamber that exposestheir tail. A heat lamp with an infrared, 250 w bulb is placed about 4inches from the tail, and left for a couple of minutes. The tail is thencleansed with an alcohol wipe and a 30 G ½ precision glide needle(Fisher Scientific), is inserted into one of the side veins forinfusion. To verify whether the needle is in the vein a small amount ofdrug is infused, if in the correct location the solution should infuseeasily without any indication of an incorrect subcutaneous location,which appears white at the site of injection. For intravenouscatheterization, Male NIH Swiss mice are anesthetized with ketamine 100mg/kg and xylazine 10 mg/kg co-administered i.p. When mice are no longerresponsive to paw pressure, the neck is shaved and prepped byalternating Betadine and alcohol wipes. Under clean conditions, a righttransverse neck incision is made and the external jugular vein isisolated. A catheter is inserted into the vein with the aid of adissecting microscope to the level of the right atrium, and is securedin the vein with nylon, 4-0 sutures and tissue adhesive, 3M Vetbond (3MAnimal Care Products, St. Paul, Minn.). The catheters are a short lengthof Tygon tubing with an inner diameter of 0.010 in. and an outerdiameter of 0.030 in. (Small Parts, Inc., Miami Lakes, Fla.). A smallincision is made in the middle of the animal's back, and a trocarinserted subcutaneously to exit at the ventral incision site. Thecatheter is then pulled through the trocar and brought out the animal'sback where it is held in place with nylon suture material and the tissueadhesive. A short piece of steel wire with a diameter of 0.011 in.(Small Parts, Inc., Miami Lakes, Fla.) is inserted into the end of thecatheter. The ventral incision is closed with 4-0 Vicryl suture materialand the mouse placed under a heat lamp for recovery. Approximately anhour later, the mouse is returned to its home cage.

Mice that survived a first administration of the combination of 0.32 mgCocE and 320 mg/kg cocaine were given this combination again, 14 daysfollowing the initial administration. All of the mice survived thissecond administration. All mice also survived a third administration 21days following the first dose combination. The effectiveness of repeatedadministration suggests that a strong immune response is not beingmounted to CocE in this preparation, perhaps because of its rapidclearance.

Results showed that a dramatic shift to the right in the potency ofcocaine in producing death was afforded by both 0.32 and 1.O mg/kgcocaine (see e.g., FIG. 18). The latency to death was markedly extendedby CocE: following 180 mg/kg i.p. cocaine, mice typically died in about3 min. After a dose of 1.0 gm/kg cocaine plus 1.0 mg of CocE, deathoccurred at 28 minutes on average. Data was also examined for theability of hBChE to block the toxicity of cocaine. At a dose of 1 mg,hBChE produced a slight increase in the dose of cocaine necessary tokill the mice (see e.g., FIG. 18). This enzyme is markedly lesseffective than CocE. Although larger doses of cocaine were necessary tokill the mice following administration of hBChE, the time to death wasnot modified by this enzyme.

Example 9 In Vivo Half-Life and Biodistribution Tests

The in vivo half-life and biodistribution of the mutant CocEpolypeptides are examined in BALB/c mice. In brief, ¹²⁵I is labeled tothe tyrosine residues of mutant CocE by using the well-establishedchloramine-T iodination method (Hunter, W M and Greenwood, F C (1962)Nature 194, 495-496). BALB/c mice are injected via their tail veins with0.1 mL of either 0.5 μCi ¹²⁵I-labeled mutant CocE or the mutant CocE-PEGconjugates. Each experimental group consists of 24 mice. Three mice aresacrificed by cervical dislocation at the 15-, 30-, 90-minute, 3-, 12-,24-, 48-, and 72-hour time intervals following the drug injection. Bloodsamples as well as tissue samples of liver, lung, heart, kidney, andspleen are collected, weighed, and measured for radioactivity using agamma counter. The blood samples are also centrifuged, and thesupernatants will be collected and counted for estimation ofplasma-associated radiolabels. The ratio of the peak to that of theinternal standard is used as the assay parameter. PK parameters arecalculated by using the KINFT (Kaltenbach, M L and Vistelle, R (1994)Anticancer Research 14, 2375-2377) nonlinear least-squares computerprogram by fitting the plasma radioactivity data to a biexponentialequation (Gibaldi, M. and Perrier, D. (1982) Pharmacokinetics):A(t)=A _(t) e ^(−k) ₁ ^(t) +A ₂ e ^(−k) ₂ ^(t)

Where A(t)=% ID/mL plasma and ID=injected dose. k₂ will be used tocalculate the first-order elimination time t_(1/2). The area under thecurve (AUC) of the plasma concentration-time curve, the steady-statevolume of distribution (Vss), total plasma clearance (CI), and the meanresidence time (MRT) are calculated from A_(I), A₂, k₁, k₂, and the bodyweight (kg) of the mouse as described by Gibaldi and Perrier, 1982). Theorgan permeability-surface area (PS) product is calculated as:PS=[V _(d) −V _(o) ]Cp _((60min))/AUC_((0-60min))

Where CP_((60min)) is the terminal plasma concentration (dpm/μL) at 60min after injection, V_(d) is the tissue volume of distributiondetermined from the ratio of disintegrations per minute per gram oftissue to Cp_((60min)), and V_(o) is the organ plasma volume. The organdelivery of the samples is determined as:% ID/g=PS×AUC_((0-60min))

Where % ID/g is the percent injected dose taken up by gram of organ.

Example 10 Immunology

CocE can be used in incomplete Freund's adjuvant (IFA) to immunize mice(see Table 4). A direct ELISA specific for CocE antibodies was set up bya standard protocol. CocE was used (1 ug/ml) to coat a 96-wellmicro-titer plate using a borate buffered saline (1.5 M NaCl, 0.5 MH3BO3, 1.0 M NaOH) to resuspend the cocaine esterase (50 uL/well). Thecoating plates was left overnight at 4 C. The coating buffer was removedthe following morning and the plates blocked with 2% normal goat serumin PBS for 1 hr at 37 C and washed 3 times. Serum from the variousgroups of mice was serially diluted in 50 μL of PBS in the wells in arange of 10² to 10′ and run in duplicate. The plates were covered andincubated for 30 minutes at 37 C. Subsequently, the plates were washed 3times and 50 μL/well of Goat anti-mouse IgG peroxidase labeled antibodydiluted 1:400. The plates were then washed 3 times and 100 μL peroxidasesubstrate solution (OPD dissolved in citratelphosphate buffer) was addedto each well. After a 5-10 minute incubation (based upon colordevelopment in the positive controls) the reaction was stopped using 3MH2SO4 (50 μL/well). The plates were read at 490 nm and titer determinedby the highest dilution that showed increases over backgroundabsorbance. Positive controls were derived by immunizing Balb/c micewith 100 μg in 100 μL of Cocaine esterase emulsified in incompleteFreund's adjuvant (IPA) by intraperitoneal (IP) injection. In Positive 1group the serum was isolated from 2 week immunized mice. In Positive 2group immunized mice were boosted using 100 μg in 100 μL by IP injectionat 2 weeks post-primary immunization and the serum collected after anadditional week (3 week post-primary).

High titers were derived from the two positive control groups immunizedwith CocE, 10⁵ and 10⁶, respectively. The antibody titers from animalsgiven CocE i.v. during cocaine challenges demonstrated detectable butrelatively low titers compared to the positive control animals immunizedby CocE plus IFA. Serum collected from animals immunized once withCocE/IFA demonstrated high titer of 10⁵, while serum from animals givenan additional boost were had a higher titer of 10⁶ (3 mice/group). Theserum collected from these animals will serve as positive controls forall subsequent titering experiments.

TABLE 4 CocE titers of immunized mice Titer (log10 Group Protocoldilution +/− SE Positive 1 (n = 3) 2 weeks Immunized (IFA/IP) 5 Positive2 (n = 3) 3 weeks Immunized/Boost 6 (IFA/IP) Challenged and Treated (3x)CocE given with cocaine 3.33 +/− 0.333 Challenged and Treated (4x) CocEgiven with cocaine 3.5 +/− 0.5 

Example 11 CocE Pegylation

Conjugation of one to two PEG polymers per enzyme molecule is generallysufficient to yield the desired protective effects (Avramis et al.(2002) Blood 99, 1986-1994). Because each wild type CocE molecule isreported to contain 8 lysine residues, with none in the active site butonly 2 being close to the active site (Turner et al. (2002) Biochemistry41, 12297-12307), targeting lysine for pegylation is unlikely toinactivate the enzyme. The mutant CocE is mixed with variousmonomethoxy-PEG (m-PEG) polymers (MW ranging from 3-12 KDa); all containan activated N-end functional hydroxysucciniyl ester group (mPEG-NHS;from Shear Water Inc., Birmingham, Ala.). PEG with a molecular weight of5.5 KDa will be first attempted, because the inventors' results andthose of other investigators (see e.g., Veronese, F M and Harris, J M(2002) Advanced Drug Delivery Reviews 54, 453-456; Avramis et al. (2002)Blood 99, 1986-1994) demonstrated that this molecular weight yields thebeneficial protection. Different molar ratios of [NH₂]:[mPEG] (theformer is calculated based on the total moles of lysine residues ofmutant CocE) ranging from 1:2 to 1:10 are tested to obtain optimalconditions. Conjugation proceeds for about 40 minutes at 4° C. withgentle agitation. The reaction products are then purified byultrafiltration (MWCO 10,000) at 4° C. Activities of the pegylatedmutant CecE products are determined by measuring the initial rates ofcocaine hydrolysis using the previously established procedure (Turner etal. (2002) Biochemistry 41, 12297-12307). In addition, MALDi-TOF massspectraphotometry is performed on these products to analyze the degreeof pegylation and their molecular weight. The pegylated products arestored at −40° C. and thawed immediately prior to their uses.

Standard characterization, including determination of the optimal pH,temperature, ionic strength, as well as kinetic parameters (e.g. Km,Vm), of the mutant CocE-PEG conjugates are conducted in PBS.Furthermore, thermal stability of the pegylated products and theirstability against proteolytic degradation are examined in the presenceof human plasma or blood. In vivo functional tests of the mutantCocE-PEG products are conducted as described above. The in vivohalf-life and biodistribution of the mutant CocE-PEG conjugates, ascompared to free CocE are conducted as described above (see Example 9).

Statistical analysis is performed on the results obtained from thepegylation experiments. Random block two-way ANOVA with Dennett'spost-test is performed on data sets with two variables using GraphPad(San Diego, Calif.) Prism for Windows and GraphPad Software. Pairedt-test is performed for experiments with two conditions.

Conjugation of mutant CocE to high molecular weight (e.g., up to 60 KDa)branched PEG can also be performed (see e.g., Reddy et al. (2002)Advanced Drug Delivery Reviews 54, 571-586).

Additionally, site specific PEGylation can be a viable alternative toreduce functional and structural heterogeneity. The removal of cysteineresidues near the active site or incorporation of cysteine residues onthe protein surface can serve as better PEGylation substrates (throughmaleimide coupling). Similarly, amine-coupling of PEG to CocE can beemployed through, for example, the conservative substitution of arginineresidues for any of mutant CocE's (nine lysines total in wild-type CocE,seven of which are surface lysines).

Preliminary findings have shown that wild-type (WT) CocE wassuccessfully linked with monomethoxy-PEG (m-PEG) polymars (MW: 5.5 KDa)containing an activated succinimidyl functional group at one end, usingthe procedures described above. A high yield of the initial CocEactivity (>70%) was recovered after the pegylation reaction. MALDI-TOFmass spectra revealed four primary peaks for the PEG-CocE products,indicating the presence of a heterogeneous mixture of the conjugatescontaining different numbers of the PEG chains (ranging from 1 to 4,respectively) (see e.g., FIG. 19). It has been suggested in theliterature that conjugation of merely 1-2 PEG chains per proteinmolecule would be able to yield PEG-induced protective effects(Veronese, F M and Harris, J M (2002b) Advanced Drug Delivery Reviews54, 457-606; Avramis et al (2002) Blood 99, 1986-1994). To this regard,the pegylation method employed here apparently satisfies such arequirement.

Example 12 RBC Encapsulation

RBC encapsulation of CocE can be accomplished via a linked PTD peptide.LMWP is selected as the PTD peptide to ferry mutant CocE into RBC,because of its potency in translocating proteins across cell membrane(Park et al. (2005) FASEB Journal, in press) and its lack of toxicity(Chang et al. (2001) AAPS Journal 3, Article #17, #18 and #19). Toensure that the encapsulated mutant CocE is permanently embedded in RBC,the linkage between CocE and LMWP can degrade automatically and rapidlyonce the mutant CocE-LMWP conjugates enter RBC. A linker such as adisulfide (S—S) bond that will be degraded quickly inside the RBC due tothe presence of elevated cytosolic glutathione and reductase activity(Trouet et al. (1982) Proceeding of the National Academy of Science 79,626-629), ensures that CocE will stay in the RBC.

To produce mutant CocE-LMWP conjugates linked with S—S bonds, the aminegroup at the N-end of LMWP (this is the only —NH₂ group on LMWP) isfirst activated with SPDP, and the activated LMWP then mixed with mutantCocE in the presence of dithiothretol (DTT) to allow for the formationof the S—S bond with one of the (four in wild-type) free cysteineresidues on mutant CocE (Turner et al. (2002) Biochemistry 41,12297-12307); according to a modified procedure previously developed(Liang et al. (2000) AAPS Pharmaceutical Science 2, Article 7). CocE isstable when being stored in DTT (Turner et al. (2002) Biochemistry 41,12297-12307), suggesting that use of these free cysteine groups forconjugation is not likely to impair the catalytic activity of thisenzyme (already performed mutation of each cysteine in wild-type CocE toa serine resulted in no dimunition of activity). The final LWMP-CocEproducts is then purified via a heparin column, and is stored bylyophilization.

Encapsulation is achieved by incubating RBC with mutant CocE-LMWP for30-60 minutes. Because PTD-mediated cell entry istemperature-independent (Schwarze et al. (1999) Science 285, 1569-1572),encapsulation is conducted at 4° C. to maximally preserve thefunctionality of RBC. The process and extent of mutant CocE entrapmentin RBC is monitored by confocal microscopy and flow cytometry analysisusing FITC-labeled mutant CocE. The morphology of mutant CocE-entrappedRBC is also be examined by SEM.

Basic characterization—including assessment of the functionality of bothRBC (e.g. oxygen-transfer activity) and mutant CocE (e.g.cocaine-hydrolyzing activity, kinetic properties such as Km, Vm, etc.),leakage of mutant CocE from RBC (i.e. by incubating mutant CocE-loadedRBC in buffer and then measuring enzyme activity in the supernatant),and stability of the entrapped mutant CocE against proteolyticdegradation—is conducted either in buffer or in plasma. Results obtainedfor the RBC-encapsulated mutant CocE are compared with those obtainedfor the free enzyme. In vivo functional tests of the mutantCocE-encapsulated RBC are conducted as described above.

Human RBC (from American Red Cross, Detroit, Mich.) are used for invitro studies. For in vivo animal studies including the functional testsand pharmacokinetic studies, however, autologous RBC from the sameanimal species are be used to avoid cell incompatibility and possibletoxic effects.

Circulation half-life (t_(1/2)) of mutant CocE-entrapped RBC isdetermined by injecting ¹²⁵I-labeled mutant CocE (i.e. prior to itsloading into RBC) into mice, according to the same procedures describedabove. Each set of experiments consists of 24 mice. Mice (3) aresacrificed at 3-, 6-, 12-, 24-hour and 3-, 6-, 10-, and 15-day timeintervals. Blood and tissue samples are collected, weighed, and measuredfor radioactivity. PK parameters including the elimination t_(1/2), aswell as tissue distribution are calculated by using the KINFT program asdescribed above. Pharmacokinetic results obtained for theRBC-encapsulated mutant CocE are compared with those obtained for freemutant CocE.

Statistical analysis is performed on the results obtained from the RBCencapsulation experiments. Random block two-way ANOVA with Dennett'spost-test is performed on data sets with two variables using GraphPad(San Diego, Calif.) Prism for Windows and GraphPad Software. Pairedt-test is performed for experiments with two conditions.

To examine if the previously suggested could fulfill the latter tworequirements, preliminary studies of PTD-mediated RBC encapsulation wereconducted using L-asparaginase as a model enzyme. LMWVP, a PTD peptidepreviously developed in Dr. Yang's laboratory (Chang et al. (2001) AAPSJournal 3, Article #17, #1, and #19) with proven, potentmembrane-penetrating activity (Park et al. (2005) FASEB Journal, inpress), was linked to asparaginase using a procedure similar to thatdescribed above. The LMWP-ASNase conjugates were then incubated with RBC(collected from DBA/2 mice) for 2 hrs at 4° C. For comparison,RBC-Ghosts containing encapsulated ASNase were also prepared accordingto the previously established procedures (Updike et al. (1976) Science193, 681-683). Preliminary results demonstrated that loading efficiencyof the LMWP-mediated method was at least comparable to, if not betterthan, the conventional, osmosis-based cell rupturing technique. A majoradvantage of the PTD-mediated method, however, is that it only requiresa single step for processing; unlike the other cell entry methods thatall require multiple steps of the loading and washing procedures.

FIG. 20 presents scanning electron microscopy (SEM) images taken fromsamples of normal RBC, ASNase-loaded RBC-Ghost, and LMWP-ASNase-loadedRBC. As seen, while the ASNase-loaded RBC from osmotic rupture/resealingmethod (i.e. RBC-Ghost) exhibited significant change in shape andmorphology, the LMWP-ASNase-loaded RBC showed virtuallyindistinguishable shape and morphology (i.e. biconcave disk) from thoseof normal RBC. These findings are in agreement with those reported bymany other investigators that PTD-rnediated cell encapsulation does notcause any significant perturbation or alteration of the cell membrane(Dietz, G P H and Bahr, M (2004) Molecular Cell Neurosciences 27,85-131; Schwarze et al. (1999) Science 285, 1569-1572; Suzuki et al.(2002) Journal of Biological Chemistry 25, 2437-2443).

To further evaluate these two RBC-encapsulation systems, preliminaryclearance studies were carried out. The half-life of ASNase activity inplasma was evaluated after intravenous injection of: (1) ASNase-loadedRBC-Ghost, and (2) LMWP-ASNase-loaded RBC. Each animal group consistedof 4 DBA-2 mice, and each mouse was given 8 units of loaded ASNaseactivity. Blood samples were withdrawn at different time intervals fromthe tail vein, and the amount of ASNase activity in the whole blood wasmeasured by direct Nesslerization of produced ammonia (Ho et al. (1970)Journal of Biological Chemistry 245, 3708-15). Results demonstrated thatthere was almost a two-fold increase in the circulation half-life forthe LMWP-ASNase-encapsulated RBC (t_(1/2): 9.2 days) compared to that(t_(1/2): 5.9 days) for the RBC-Ghost (see e.g., FIG. 21). It iscurrently unknown what the difference between the half-lives of theencapsulated RBC and the normal, untreated RBC. Nonetheless, theseresults of 2-fold increase of t_(1/2) over RBC-Ghost demonstrate themerit of this approach, as it was reported in the literature that evenby utilizing the RBC-Ghost encapsulation method, the in vivo ASNaseactivity had already been prolonged from 26 hours for the free ASNase to29 days for the RBC-Ghost-encapsulated ASNase (Kravtzoff et al. (1996)European Journal of Clinical Pharmacology 49, 465-470): already a10-fold increase. Hence, another 2-fold increase of this t_(1/2) by theencapsulation method described herein is particularly effective.

To confirm that the RBC-encapsulated ASNase could still retain itsoriginal biological functions, anti-tumor effects by both theRBC-Ghost-ASNase and the RBC-ASNase were examined on tumor-bearing mice.L5178Y mouse lymphoma cells were cultured, and to each DBA/2 mouse 7×10⁵cancer cells were injected intraperitonially. Five days after tumorimplantation, mice with similar bodyweights were selected and dividedinto three groups: (1) Control group given saline only; (2)ASNase-loaded RBC-Ghost; and (3) LMWP-ASNase-loaded RBC. Each groupconsisted of 5 mice, and each experimental mouse was given 0.1 mL of thedrug-encapsulated RBC (or Ghost). Results showed that the averagesurvival times for the untreated control, ASNase-RBC-Ghost-treated, andLMWP-ASNase-RBC-treated groups were 10.0, 12.6, and 14.4 days,respectively (see e.g., FIG. 22). It should be noted that although thesurvival time among the three groups differed by only about 2 days, theeffects of the treatment by the RBC-encapsulated ASNase was still quitedramatic; considering the fact that only 0.1 mL of the RBC suspension,which was equivalent to merely 5% of the total blood volume of themouse, was given to each mouse for the anti-tumor treatment. Overall,these findings with the ASNase model enzyme demonstrated that theRBC-encapsulated enzyme was still therapeutically active. It is notedthat cocaine is more permeable across the RBC membrane than theasparaginase substrate.

Thus, utilization of RBC-encapsulated CocE in treating cocaine-relatedconditions is an effective approach because cocaine readily crosses theRBC membrane (and actually somewhat concentrates in RBC) (Javaid et al.(1978) Journal of Chromatography 15, 105-113), PTD-mediatedencapsulation does not alter the physical and/or chemical properties ofthe RBC, and the RBC-encapsulated enzyme functions as if free.

Example 13 Endotoxin Removal

Endotoxin contamination of CocE mutants can be performed in severalways. Ideally, endotoxin decontamination decreases the concentration tolevels less than 10 EU/mg protein. Methods of decontamination includealternative ion-exchange column chromatography conditions, sizeexclusion, polyethyleneimine (PEI) and hydrophobic columnchromatography, ultrafiltration and detergent extraction. An endotoxindetection system (PYROGENT 5000, Cambrex) is used to determine theendotoxin content of preparations. The assay is based on the anti-LPSfactor from Limulus amebocyte lysate (LAL). The sensitivity of the assayis between 0.01 and 100 EU/ml, well within the levels required. Thespectrophotometric assay is designed in a 96-well microtiter-plateformat. Fractionation of endotoxin and CocE and/or mutant CocE by thefollowing procedures can be assayed for both endotoxin level and cocaineesterase activity. Cocaine esterase activity is measured utilizing aspectrophotometric assay that takes advantage of the intrinsicabsorption of cocaine at 240 nm. Upon hydrolysis, the absorption spectrareveals a dramatic reduction in the 240 nm peak (Turner et al. (2002)Biochemistry 41, 12297-12307).

Anion Exchange Chromatography: Current conditions involve the use offast performance liquid chromatography (FPLC) on a Q-Sepharose column atpH 8.0 for endotoxin decontamination. The buffer conditions (pH) can beoptimized to maximize the both the adsorption of CocE and/or mutant CocEand separation of endotoxin. Fluted fractions are assessed by activitymeasure of CocE or mutant CocE activity (spectroscopic assay of thehydrolysis of cocaine, absorption at 240 nm). Endotoxin levels areassessed using PYROGENT 5000 (above).

Size Exclusion Chromatography and Ultrafiltration: Endotoxin can existas monomeric forms (MW˜1-2×10⁴) or in micellar form (MW˜4×10⁶ to 1×10⁶),depending on the buffer conditions. The presence of detergents such ascholate favors the monomeric form whereas divalent cations (e.g. Ca²⁺)favor the micellar form (Hirayama, C and Sakata, M (2002) Journal ofChromatography B Analytical Technology Biomedical Life Science 781,419-432). This property of endotoxin is used to separate the micellar(i.e. in the presence of divalent cations such as Mg²⁺ or Ca²⁺) fromCocE and/or mutant CocE by Gel filtration chromatography using either aSephadex 75 or Sephadex 200 (Pharmacia) column resin. Micellar endotoxinshould not be retained on the column and should pass through the voidwhereas CocE and/or mutant CocE should elute as a monodispersed proteincorresponding to a 65 kDa protein. Similarly, the capacity ofultrafiltration units to separate the micellar forms of endotoxin fromCocE and/or mutant CocE will be assessed. Ultrafiltration units are nowavailable with molecular weight cutoffs of 3×10⁵ to 1×10⁶, well withinthe range required to retain micellar endotoxin but not CocE itself.

Polyethyleneimine Chromatography: Mitzner et al. (1993) and Morimoto etal. (1985) have used PEI-immobilized on cellulose beads or on cellulosefiber, respectively to remove endotoxin from BSA preparations.PEI-chromatography column is a very weak anion exchanger that may infact take advantage of some hydrophobic properties of endotoxin and thuspreferentially adsorb it. Various PEI-silica bead preparations areavailable from Sigma, depending on the bead size. While silica-basedbeads are more classically associated with HPLC applications we willpack columns for lower pressure work and will select the 200 um meshsizes.

Triton X-114 Extraction: Triton X-114 phase separation has beensuccessfully used to separate endotoxin from albumin and catalase (Aida,Y and Pabst, M J (1990) J Immunological Methods 132, 191-195). Adia andPabst report a 1000-fold decrease in endotoxin concentration following asingle Triton X-114 extraction step. CocE and mutant CocE samples areincubated with equal volumes of Triton 114 and allowed to incubate firston ice and then at 37° C. for 15 minutes. The Triton X-114 phase thatcontains the endotoxin is removed by centrifugation. As noted earlier ithas been demonstrated that CocE is considerably thermal labile, but maybe protected somewhat by the presence of the substrate, cocaine. Ifincubation of CocE with Triton X-114 at 37° C. results in significantCocE inactivation, the enzyme will be stabilized by including excesssubstrate during the extraction.

Example 14 Mutagenesis of the CocE Host Organism

There exists several bacterial strains that exhibit temperaturesensitive growth on cocaine esterase byproducts, including severalPseudomonas strains, and these organisms can be adapted for temperaturesensitive growth on cocaine by addition of the CocE gene encoded withinsuitable plasmid vectors. For example, the CocE gene was originallysequenced from Rhodococcus MB1 by subcloning gene fragments intoRhodococcus erythropolis CW25, a bacterium unable to metabolize cocainebut able to grow on the cocaine esterase byproducts ecgonine methylester and benzoate (Bresler et al. (2000) Applied & EnvironmentalMicrobiology 66, 904-908). The CocE gene has been subcloned into shuttlevectors pJAK-14 and pMMB67EH (see e.g., Example 1). These plasmids arecapable of expression in any gram-negative bacterium includingPseudomonas, and additionally the pMMB67EH plasmid enables high levelsof expression and ease of transformation by bacterial conjugation withthe use of helper plasmid pRK2013. Transformation of either plasmid intobacteria that exhibit temperature sensitive growth on cocaine esterasebyproducts (e.g., Pseudomonas strains), enables temperature sensitivegrowth on plates containing cocaine as a sole source of carbon.Mutagenesis of both the original Rhodococcus MB1 and the CocEplasmid-containing Pseudomonas strains is performed using UV-lightradiation at 260 nm. Exposure is titrated such that 90-95% of cells arekilled. The remaining cells are recovered in nutrient media for 1 hr at26° C., collected and enriched in the presence of cocaine as previouslydescribed (Britt, et al. (1992) Journal of Bacteriology 174, 2087-2094).Enrichment is performed at 37° C. to select for thermostable variants ofthe CocE. Finally, cells are plated onto minimal media agar platescontaining 10 mM cocaine and incubated at 37° C. Single colonies aregrown and tested for CocE activity at 37° C. PCR amplification of theCocE gene is performed on mutants found to produce active and solubleCocE at 37° C., and the amplification products are subcloned into thepET-22B(+) plasmid for further characterization. As a prelude to CocEmutagenesis, it may be necessary to mutagenize the native Pseudomonasstrains in order to preselect for no temperature sensitivity at 37° C.when grown on cocaine hydrolysis products, and then demonstratetemperature sensitivity on cocaine at 37° C. after cloning in CocE.

Example 15 High Throughput Screening Method for Identification ofThermostable CocE Mutants

Several high throughput screening methods for identification ofthermostable CocE variants were implemented. Because the wild-typeenzyme is known to have thermal-instability at temperatures above 30°C., after transformation into E. coli BL21 cells, colonies aresubcultured and protein expression induced at 16° C. Expressed proteinsare then tested for esterase activity at temperatures 30° C. and above.After several rounds of mutagenesis and testing at increasingtemperatures thermostable mutants are achieved. Each individual mutantCocE is then prepared, purified, and tested for activity andthermostability at 37° C., as described above (see Examples 1 and 4)

Bacterial colonies containing mutant polypeptides are screened fromdirectly from agar plates by nitrocellulose filter imprint of replicaplates followed by lysis of the bacteria and protein fixation.Determination of enzymatic activity at various temperatures is achievedby monitoring the accumulation of benzoic acid, the acidic by product of(−)cocaine. A moist nitrocellulose imprint is placed onto a dried filterpaper previously saturated with a mixture of cocaine at pH 7.4 with nobuffer and a pH indicator that transitions from colorless to color uponacidification e.g., methyl red. Active enzyme is identified by colorchange and colonies are appropriately harvested. The detection methodbased on acidification through the formation of benzoic acid is employedfor the detection of cell expression of catalytic antibodies thathydrolyze cocaine at the benzoyl ester group, the same site cleaved byCocE. Alternatively, detection by nitrocellulose imprint is achieved byexposure to a cocaine thiol-derivative and subsequent detection ofsulfhydryl groups via a precipitating heavy-metal (e.g., mercury-based)indicator system.

Bacterial colonies containing mutant polypeptides are also screened bysubculturing into liquid medium and testing directly for cocaineesterase activity using a cocaine thiol-derivative and detection usingthe colorimetric thiol indicator, Ellman's reagent (see e.g., FIG. 6).Ellman's reagent rapidly forms a disulfide bond with free thiol-groupsand releases a colored thiolate ion which absorbs at 412 nm. Culturesincubated overnight at 16° C. in the presence of IPTG to induce proteinexpression (20 l) are mixed with 1 mM benzoylthioester cocainederivative, and 500 uM Ellman's reagent in 100 mM sodium phosphate pH7.4 to a final volume of 200 l. Results (see e.g., FIG. 6) indicate thatcells containing the wild-type CocE enzyme are able to cleave thebenzoylthioester at much higher levels than cells alone.

Finally bacterial colonies containing mutant polypeptides are screenedby subculturing into liquid medium followed by lysis and isolation ofmutant polypeptides using an affinity medium. For example, lysed cellsare washed through nickel-agarose filter-plates allowing collection andsubsequent elution of 6×HIS tagged proteins (for example, Ni-NTASuperflow 96-Bio-robot kit (Qiagen). Alternatively, cells are lysedwithin nickel-coated microtiter plates allowing binding of 6×HIS fusionproteins and subsequent removal of contaminants (for example,immobilizer nickel chelate plates (Nunc) or NEN Nickel-chelate flashplates (Perkin Elmer)). Similarly, lysed cells are incubated with nickelcoated microbeads (such as Ni-NTA magnetic agarose beads (Qiagen)),followed by subsequent removal of contaminating proteins. Isolatedesterase protein activity are then tested using any of the assayspreviously mentioned (such as the spectrophotometric activity assay(Examples 1 and 4), the tritiated cocaine activity assay (Example 1),the benzoic acid pH indication activity assay, the thiol-derivativecocaine detection systems, the use of cocaine aptamers (Stojanovic, M.N., de Prada, P. & Landry, D. W. (2001) J Am Chem Soc 123, 4928-31;Stojanovic, M. N. & Landry, D. W. (2002) J Am Chem Soc 124, 9678-9) bymonitoring changes in fluorescence upon degradation of cocaine, or bythe use of a generic esterase substrate such as 4-nitrophenyl acetateand monitoring of colorimetric changes at 420 nm as described previously(Halgasova, N. et al. (1994) Biochem J 298 Pt 3, 751-5; O'Conner, C. J.& Manuel, R. D. (1993) J Dairy Sci. 76:3674-3682).

Example 16 Preliminary Analysis of N197K Mutant Polypeptide

Preliminary analysis of the N197K mutant polypeptide (SEQ ID NO: 42)showed good stability after 1 hour at 37° C. on day 0. Vmax and Kmvalues are shown in Table 5. A recheck on day 3 showed similarstability. Vmax and Km values are shown in Table 6. Higher Km values onday 3 were due to reuse of old cocaine. Gel filtration of samplesincubated at 37° C. showed formation of aggregates.

TABLE 5 N197K initial test (Day 0) Equation 1 Best-fit values N197K (0)N197K (60) VMAX 2928 2187 KM 34.77 24.24

TABLE 6 N197K repeat test (Day 3) Equation 1 Best-fit values N197K (0)N197K (60) VMAX 3357 3085 KM 102.2 113.5

Example 17 Identification of Thermally Stable Mutant CocE Polypeptides

Thermally stable mutant CocE polypeptides were identified by determining_(1/2) of the mutant CocE polypeptides. Briefly, enzymes werepreincubated at 37° C. for varying times. Activity measurements weredetermined at RT (25° C.). Mutant enzymes with _(1/2) of greater than 12minutes (the _(1/2) of wild type CocE) were considered thermally stable(See, e.g., FIG. 27 and Table 7). As demonstrated by the combinationmutant CocE polypeptide T172R/G173Q, in various embodiments, thecombination of two single mutations with lower or no stability canresult in a thermally stable combination.

TABLE 7 Thermally stable mutant CocE polypeptides Mutant Stability @ 37°C. (t_(1/2)) T122A No Q123E No S159A No S140A No S167A/W52L No T172R ~46min V121D No L163V No F189A No F189A/T172R ~40 min (Similar to T172R)C107S No W220A No F189L No A193D No T172R/A193D ~40 min (Similar toT172R) G173Q ~25 min T254R No N42V No T172R/G173Q ~326 min G171Q/T172R/G173Q No G171A No G173A No wt-I175-G-D185 Nowt-T176-G-G-D185 No T172R/G173Q-I175-G-D185 T172R/G173Q-I175-G-G- ~75min A186 T172R/G173Q-T176-G-G- ~75 min D185 S177Q No D45R No F47R NoL169K ~274 min  L174R No A181K No S179R No F189K   25 min V190K No A194KNo R182K No

Example 18 Protection of Aggregation of Thermal Stable Cocaine EsteraseMutants

Protection from aggregation of thermal stable CocE mutants was assessedusing size exclusion chromatography. Briefly, enzymes were preincubatedat 37° C. for 0 minutes or 60 minutes and resolved by size exclusionchromatography. Results for wild-type CocE, T172R and T172R/G173Q areshown in FIG. 28.

Example 19 Low-UV Spectra

Low-UV CD spectra data were obtained using an Aviv SpectropolarimeterMode 400, with the help of Norma Greenfield, UMDNJ, using a 5-cellholder and 0.2 mg/ml protein concentrations. Raw data values obtainedwere blanked on control PBS, smoothed, and deconvoluted using the CCAalgorithm as described by A. Perczel, K. Park, and G. D. Fasman,[Analysis of the circular dichroism spectrum of proteins using theconvex constraint algorithm: a practical guide. Analytical Biochemistry203, 83-93 (1992).] This algorithm finds the minimum number of curvesneeded to reconstruct a data set, and expresses the percentage eachcurve contributes to the data set as a function of temperature.

Below is presented a low UV CCA denconvolution analysis of CocE WT and 4mutants, obtained in a single CD spectra melt using a 5-cell holder. Theexperiment was performed over the period of 8 hours, from 0-80° C.

Temperature dependent melting was observed between the wavelengthstested (200-250 nm), and the smoothed spectra for each mutant are shownin FIG. 29. Deconvolution via the CCA algorithm indicated that eachspectra was best described by a set of three curves, as shown in FIG.30. This suggests that the melting of CocE is at least a two stepprocess, moving from an original curve (curve 1) to an intermediateunfolding step (curve 2), and finally fully denatured protein (curve 3).The % contribution that each temperature played in describing thesethree curves is shown in FIG. 31. Dose response analysis was used toapproximate the temperature at which the initial spectra melts (1), theformation and melt of the intermediate state (2), and the accumulationof the fully melted protein (3). These numbers were collected andplotted in FIG. 32 and shown in Table 8.

TABLE 8 Melting points of each step #1 melt #2 formation #2 melt #3formation

 WT 37.04 34.57 50.98 59.86

 T172R 39.22 38.11 54.56 56.85

 T172R-A193D 38.11 46.47 47.68 58.72

 T172R-F189K 38.8 46.81 48.42 57.23

 T172R-G173Q 40.62 45.61 46.17 56.61

The most thermostable mutant T172R-G173Q (as determined in other assays)showed the highest temperature melting of the original curve 1 (40° C.vs 37° C. for wild-type), and the lowest temperature for bothdisappearance of the curve 2 intermediate (46° C. vs 50° C. forwild-type) and appearance of the fully melted curve 3 (56° C. vs 59° C.for wild-type).

In summary, it appears that all mutants undergo a 2-step meltingprocess.

Example 20 Stabilization Using Products and Inhibitors

Cocaine esterase (CocE) cleaves cocaine to produce Benzoic acid andEcgonine methyl ester. Briefly, alternative substrates and inhibitors ofcocaine, as well as compounds able to thermostabilize the enzyme, wereinvestigated generally by substituting amide and thiol analogues at thereactive ester bond, or removing the bond (for inhibitors), substitutingbenzoyl analogues in place of the benzoic acid leaving group, and/orremoving or altering the methyl-ester group on the ecgonine portion ofthe molecule. As discussed below, it was determined that somesubstrates, products, and inhibitors stabilized thermal denaturation ofwild type CocE, as well as prevented thermally-induced aggregation ingel electrophoresis.

Cocaine is the natural substrate of Cocaine esterase (CocE). Cocainecleavage was monitored by a drop in absorbance at 240 nm. Cocaine (mMrange) prevented 37° C.-induced formation of high molecular weight CocEaggregates (0.1 mg/ml enzyme concentrations) (see, e.g., FIG. 33).Cocaine (uM quantities) stabilized 37° C.-induced activity loss (see,e.g., FIG. 34), although the mechanism of this stabilization iscomplicated due to substrate inhibition at higher concentrations.

Benzoic acid is the natural product of CocE and a weak inhibitor of CocEcleavage of 4-nitrophenyl acetate (Ki 310 uM). Benzoic acid prevented37° C.-induced formation of high molecular weight CocE aggregates (0.1mg/ml enzyme concentrations) (see, e.g., FIG. 35). Benzoic acid (uMquantities) stabilized 37° C.-induced activity loss (see, e.g., FIG.36), although the mechanism of this stabilization is complicated due tosubstrate inhibition at higher concentrations.

CocE catalyzes the cleavage of 4-nitrophenyl acetate (4NPA) to4-nitrophenol (4NP) and acetate. The cleavage reaction was monitored byobservation of product formation at 400 nm. Both 4NP and 4NPA (mM range)prevented 37° C.-induced formation of high molecular weight CocEaggregates (0.1 mg/ml enzyme concentrations).

Phenylboronic acid is a potent inhibitor of CocE (Ki 250 nM).Phenylboronic acid stabilized 37° C.-induced aggregation of CocE with anEC50 of 0.2 UM by densitometry analysis.

Using the above, a screening assay is developed to screen for smallmolecules that might similarly stabilize the enzyme but not necessarilyoccupy the active site. Molecules identified as stabilizing moleculesare used to stabilize the proteins disclosed herein until ready for use.

The invention claimed is:
 1. An isolated mutant cocaine esterase (CocE)polypeptide comprising an amino acid sequence at least 90% identical toSEQ ID NO: 1, having at least one substitution, addition or deletion,wherein the at least one substitution, addition or deletion is selectedfrom the group consisting of L163V; V225I; I218L; A310D; A149S; S159A;S265A; S56G; W220A; S140A; F189L; A193D; T254R; N42V; V262L; L508G;Y152H; V160A; T172R; Y532F; T74S; W285T; L146P; D533S; A194R; G173Q;C477T; K531A; R41I; L119A; K46A; F84Y; T172R-G173Q; L169K; F189A; N197K;R182K; F189K; V190K; Q191K; A194K; I175-G-G-A186; and T176-G-G-D185; andthe mutant CocE polypeptide has esterase activity.
 2. The mutant CocEpolypeptide of claim 1, wherein the mutant CocE polypeptide comprisesincreased thermostability at 37° C. as compared to wild-type CocE havingthe amino acid sequence of SEQ ID NO:1.
 3. The mutant CocE polypeptideof claim 2, wherein the increase in thermostability of the mutant CocEpolypeptide over wild-type CocE having the amino acid sequence of SEQ IDNO:1 is about 2.1 kcal/mol or greater.
 4. The mutant CocE polypeptide ofclaim 1, wherein at least two amino acid residues are substituted. 5.The mutant CocE polypeptide of claim 1, wherein at least three aminoacid residues are substituted.
 6. The mutant CocE polypeptide of claim1, wherein at least four amino acid residues are substituted.
 7. Themutant CocE polypeptide of claim 1, wherein at least five amino acidresidues are substituted.
 8. The mutant CocE polypeptide of claim 1,comprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 3 (L163V); SEQ ID NO: 7(V225I); SEQ ID NO: 8 (I218L); SEQ IDNO: 9 (A310D); SEQ ID NO: 10 (A149S); SEQ ID NO: 11 (S159A); SEQ ID NO:12 (S265A); SEQ ID NO: 13 (S56G); SEQ ID NO: 14(W220A); SEQ ID NO: 16(S140A); SEQ ID NO: 17 (F189L); SEQ ID NO: 18 (A193D); SEQ ID NO: 19(T254R); SEQ ID NO: 20 (N42V); SEQ ID NO: 21 (V262L); SEQ ID NO: 22(L508G); SEQ ID NO: 23 (Y152H); SEQ ID NO: 24 (V160A); SEQ ID NO:25(T172R); SEQ ID NO: 26 (Y532F); SEQ ID NO: 27 (T74S); SEQ ID NO: 28(W285T); SEQ ID NO: 29 (L146P); SEQ ID NO: 30 (D533S); SEQ ID NO: 31(A194R); SEQ ID NO: 32 (G173Q); SEQ ID NO: 33 (C477T); SEQ ID NO: 34(K531A); SEQ ID NO: 35(R41I); SEQ ID NO: 36 (L119A); SEQ ID NO: 37(K46A); SEQ ID NO: 38 (F84Y), SEQ ID NO: 39 (T172R-G173Q); SEQ ID NO: 40(L169K); SEQ ID NO: 41 (F189A), SEQ ID NO: 42 (N197K), SEQ ID NO: 43(R182K), SEQ ID NO: 44 (F189K), SEQ ID NO: 45(V190K), SEQ ID NO: 46(Q191K), and SEQ ID NO: 47 (A194K).
 9. The mutant CocE polypeptide ofclaim 1, encoded by a nucleic acid having at least 90% sequence identitywith SEQ ID NO:
 2. 10. The mutant CocE polypeptide of claim 9,comprising a substitution, addition or deletion of F189A/T172R;T172R/A193D; T172R/G173Q; T172R/G173Q-I175-G-G-A186; orT172R/G173Q-T176-G-G-D185.
 11. The polypeptide of claim 1, wherein themutant CocE polypeptide has at least or about 10% of the esteraseactivity of wild-type CocE polypeptide.
 12. The mutant CocE polypeptideof claim 1, wherein the mutant CocE polypeptide is pegylated.
 13. Themutant CocE polypeptide of claim 1, stabilized by a substrate orinhibitor.
 14. The mutant CocE polypeptide of claim 1, comprising asubstitution, addition or deletion of T172R; F189A; A193D; G173Q;G173Q-I175-G-G-A186; G173Q-T176-G-G-D185; L169K; or F189K.
 15. Apharmaceutical composition comprising the mutant CocE polypeptide ofclaim 1 and a pharmaceutically acceptable carrier or excipient.
 16. Thepharmaceutical composition of claim 15, suitable for parenteraladministration.
 17. The pharmaceutical composition of claim 15, whereinthe mutant CocE polypeptide comprises a lyophilized mutant CocEpolypeptide.
 18. The pharmaceutical composition of claim 15, wherein thepharmaceutical composition is a lyophilized pharmaceutical composition.19. The mutant CocE polypeptide of claim 1, comprising substitutions ofT172R and G173Q.